Recombinant CCN domain proteins and fusion proteins

Abstract

The present invention relates to recombinant proteins having an amino acid sequence corresponding to or related to the thrombospondin type I repeat homology domain of a member of the CCN family of proteins, and uses thereof. The present invention also relates to fusion proteins comprising an amino acid sequence corresponding to or related to the thrombospondin type I repeat homology domain of a member of the CCN family of proteins, combined with a fusion partner and, optionally, a linker region. Also disclosed herein are novel protease-resistant Fc fragments.

Description

[Technical field]

[0001] The present invention relates to recombinant proteins having amino acid sequences corresponding to or related to the thrombospondin type 1 repeat homology domain (domain III) of a member of the CCN family of proteins, and uses thereof, particularly including such proteins that are truncated and / or contain specific amino acid modifications. Furthermore, the present invention relates to fusion proteins comprising an amino acid sequence corresponding to or related to the thrombospondin type 1 repeat homology domain of a member of the CCN family of proteins, optionally combined with a fusion partner, optionally through a linker peptide. In particular, the fusion partner is a monomeric protein, and the fusion protein is monomeric.

[0002] Also disclosed herein are novel protease-resistant Fc fragments. [Background technology]

[0003] CCN proteins are a family of secreted glycoproteins. CCN was originally identified as the first three members of the gene family; C yr61, C TGF and NThe name was created as an acronym derived from OV. However, this acronym has been applied in recent years as a contraction of Cellular Communication Network Factors and has been approved by the HUGO Gene Nomenclature Committee (Perbal B, Tweedie S and Bruford E, J Cell Commun Signal. 2019 Sep;13(3):435). The protein is often classified as a matricellular protein associated with the extracellular matrix (ECM). CCN proteins are not part of the scaffolding function that organizes cells into tissues, but rather are signaling proteins and may function as independent autocrine or paracrine signaling factors or as modifiers of other extracellular signaling proteins. Together with a set of three Wnt-inducible signaling pathway proteins (WISP1 / CCN4, WISP2 / CCN5, and WISP3 / CCN6), they comprise a family of six homologous cysteine-rich proteins in mammals, renamed CCN1-6.

[0004] The first members of the CCN family share a modular structure, with an N-terminal peptide signal for secretion followed by four conserved domains. The first domain shows sequence homology to insulin-like growth factor binding proteins (IGFBPs) and is therefore known as the IGF-binding protein homology domain, but has negligible affinity for IGFs. The second domain is known as the von Willebrand factor C-type repeat (VWC) homology domain, which is often found in extracellular matrix (ECM) proteins. The third domain is known as the thrombospondin type I repeat homology domain (TSP-1), which may be involved in the attachment of CCN proteins to integrins. The fourth domain is a cysteine-rich C-terminal repeat or cystine knot homology domain, a domain that has been reported to bind heparin. The fifth member of the CCN protein family, WISP2 (Wnt1-inducible signaling pathway protein 2), also known as CCN5, is atypical in that it lacks the carboxyl-terminal cystine-knot domain (domain 4). The TSP-1 homology domains of the CCN protein family share 34% amino acid sequence identity and 25% sequence similarity (as analyzed by ClustalOmega; see below for reference). The four-domain CCN proteins contain 38 conserved cysteines throughout the primary sequence, except for CCN6, in which the four cysteines in the VWC homology domain are not conserved among other family members. For CCN5, which also lacks the carboxyl-terminal cystine-knot homology domain, all cysteines in the IGFBP, VWC, and TSP-1 homology domains are conserved compared to other CCN family members.

[0005] A non-conserved, protease-sensitive central region, often referred to as the hinge region, separates the two halves of the protein. Expression of CCN proteins is regulated at the transcriptional, post-transcriptional and translational levels in response to changes in environmental stimuli.

[0006] Information regarding the domain organization of the CCN protein family can be found, for example, in Liu et al., 2017, Journal of Diabetes, 9, pp. 462-474. At the cellular level, CCN proteins may have diverse regulatory roles at the interface of the extracellular matrix and the cell surface. CCN proteins may control cell adhesion, migration, proliferation, differentiation, apoptosis, survival, senescence and gene expression. By regulating one or more aspects of these cellular functions in a cell type-specific manner, CCNs coordinate complex biological processes, including cardiovascular and skeletal development during embryogenesis, and inflammation, wound healing, and tissue injury and repair in adults. Generally speaking, the four domains CCN1-4 and CCN6 (especially CCN1, CCN2 and CCN4) may exert profibrotic activity, whereas CCN5, which contains only three domains I-III, has antifibrotic activity.

[0007] CCN proteins are also involved in a wide variety of pathological conditions, such as organ failure due to progressive fibrosis, for example liver fibrosis and idiopathic pulmonary fibrosis, as well as cancer invasion and metastasis.In this regard, see Jun and Lau, 2011, Nat.Rev.Drug.Discovery, 10(12), pp.945-963.Again, generally speaking, the four-domain CCN proteins, and in particular CCN2, have been shown to be involved in the mechanisms of a variety of fibrotic diseases, while in preclinical disease models of these diseases, it has been shown that increasing CCN5 levels, conversely, can be beneficial.

[0008] Kaasboell et al., J. Biol. Chem, 293:46, pp. 17953-17970, report that connective tissue growth factor (CTGF), also known as CCN2, is synthesized and secreted as an inactive preproprotein that requires proteolytic cleavage to release biologically active CCN2, and that a homodimer of a C-terminal fragment composed of domains III-IV represents the fully biologically active form of CCN2, and finally that the major reported activity of CCN2 can be reproduced by a homodimer of the C-terminal domains III-IV fragment. Activity assays reported by Kaasboell et al. revealed that neither the unprocessed full-length CCN2 nor the N-terminal fragment composed of domains I-II was biologically active. Furthermore, proteolytic processing of full-length CCN2 by matrix metalloproteinase (MMP) activity was found to release its latent activity. Taken together, the findings reported by Kaasboell et al. indicate that prepro-CCN2 is autoinhibited by N-terminal domains I and II. It was also found that C-terminal domains III and IV fragments of CCN1 and CCN3 are sufficient to activate rapid cell signaling and induce cellular physiological responses. However, it is unknown what degree of endopeptidase cleavage of the hinge region of CCN1 and / or CCN3, or any other CCN protein family members, is required for the release of biological activity.

[0009] CCN2 is known to be highly expressed during development, in a variety of pathological conditions with enhanced fibrosis and tissue fibrosis, and in some cancers (Jun and Lau, 2011, supra). The fact that CCN proteins are involved in a wide variety of pathological conditions, are extracellular proteins mechanistically involved in the development of fibrosis, and show only limited expression in healthy organisms makes them attractive as therapeutic targets.

[0010] Jeong et al., 2016, J. American College of Cardiology, 67:13, pp.1557-1568, report on a study that investigated the role of adeno-associated virus-mediated gene transfer of CCN5 into murine hearts after experimentally induced cardiac pressure overload. The study concludes that CCN5 may reverse established cardiac fibrosis by inhibiting myofibroblast generation and promoting apoptosis of the cells in the myocardium, suggesting that CCN5 may provide a platform for developing anti-cardiac fibrosis therapies.

[0011] In US2008 / 0207489 a method for treating disorders based on smooth muscle proliferation is disclosed, which involves expression of CCN5 or administration of CCN5 protein to smooth muscle cells.

[0012] In EP2556839, a composition is provided that includes a gene carrier that includes a nucleotide sequence encoding full-length CCN5 or CCN2ΔCT, and its role in the treatment of heart failure is suggested. CCN2ΔCT in EP2556839 is defined as the amino acid sequence of CCN2 truncated after K251 (uniprot numbering).

[0013] Although overexpression of CCN5 has been reported in several experimental systems, resulting in phenotypes opposite to those of CCN2 overexpression (Jeong et al., supra; Yoon et al., J Mol Cell Cardiol, 49(2), 294-303 Aug 2010), antagonism of four-domain CCN proteins by CCN5 has not been reported to the best of our knowledge. Notably, the structural basis of CCN5 / WISP2-mediated antagonism of other CCN family members was unknown prior to the work presented in this invention.

[0014] We have shown earlier that full-length CCN2 (FL-CCN2) is a preproprotein, an inactive precursor, and that a fragment containing domains III and IV appears to convey all biologically relevant activities of CCN2. It remains unknown how much CCN protein is generally secreted as an inactive preproprotein that requires proteolytic activation. However, the susceptibility of the full-length CCN protein to numerous proteases, as demonstrated by the present inventors (Kaasbоell et al., J. Biol. Chem. (2018) 293(46)17953-17970) and other researchers (Butler, GS et al. Matrix Biol 59, 23-38(2017) and Guillon-Munos, A. et al. J Biol Chem 286, 25505-25518(2011)), indicates that the unmodified full-length CCN protein would be highly unsuitable as a drug for stability reasons, both during recombinant protein production and after in vivo administration. This inappropriateness of using unmodified full-length CCN proteins as therapeutic proteins also applies to fusion proteins of full-length CCN proteins, as described for example for full-length CCN1 (Schutze, N. et al. (2005) Protein Expr Purif 42, 219-225) and full-length CCN6 (Schutze, N. et al. (2007) BMC Cell Biol 8, 45). It is well known in the field of CCN proteins that the susceptibility of these proteins to proteolysis is one of the reasons why it is very difficult to produce recombinant CCN proteins. Furthermore, based on the novel findings of Kaasboell et al. (J Biol Chem 2018; 293 (46): 17953-17970), recombinant full-length CCN proteins may be far from ideal biological drugs because their activity may depend on being previously proteolytically processed, which makes their pharmacokinetics and pharmacodynamics unpredictable.Furthermore, in the case of Fc-fusion proteins, it has also been shown that the arrangement of components, such as the peptide linker, CCN fragment and Fc fragment, as well as their proteolytic susceptibility, is important for the efficacy and potency of the recombinant fusion protein. An example of this is in a paper published by the present inventors (Kaasboell et al. (2018)), in which mutants of Fc-fusion proteins containing domains III-IV of CCN2 were found to have highly diverse activities in a manner that was not easily predictable in advance.

[0015] It has been reported that the action of CCN proteins is sensitive to antagonism by high concentrations of synthetic peptides derived from the primary sequence of CCN proteins. One example is the inhibition of recombinant CCN2-stimulated AKT phosphorylation in Rat2 fibroblasts by a peptide derived from the IGFBP homology domain, domain I, and to a lesser extent, by a peptide derived from the TSP-1 repeat homology domain, domain III, of CCN2 (Moe et al., J. Cell Commun. Signal. (2013) 7:31-47). Another example is the inhibition of CCN2 (domain IV)-stimulated adhesion of hepatic stellate cells by a peptide derived from the cystine-knot homology domain, domain IV of CCN2 (Gao R and Brigstock DR., J Biol Chem. 2004 Mar 5;279(10):8848-55). Furthermore, peptides derived from domain III of CCN1 (Leu et al. J. Biol. Chem, 2003, Vol. 278, No. 36, Issue of September 5, pp. 33801-33808, 2003) and domain III of CCN1, CCN2, CCN3, CCN5 and CCN6 (Karagiannis EG and Popel The International Journal of Biochemistry & Cell Biology 39 (2007) 2314-2323) have shown some antiangiogenic effects in in vitro assays with HUVEC cells (Leu et al. J. Biol. Chem, 2003, and Karagiannis EG and Popel, Int J Biochem Cell Biol 39 (2007)) and anti-adhesive effects on 1064SK human foreskin fibroblasts (Leu et al. J. Biol. Chem, 2003), but these peptides contain only one (Leu et al. J. Biol. Chem, 2003) or two (Karagiannis EG and Popel, Int J Biochem Cell Biol 39, 2007) of the central conserved cysteines of the invention described in this document.Cysteines in domain III of CCN protein are known to generate disulfide bridges, as evidenced in CCN2 endogenously expressed from HUVEC cells (Lu, S et al. (2015) Nat methods 12, 329-331) and purified recombinant CCN2 (Kaasboell et al., J. Biol. Chem. 2018). The disulfide bridges shown in CCN2, spanning C199 to C228 (uniprot numbering), give the amino acid chain a complex 3D structure that folds over itself. This indicates that it is not possible to expect the complete domain III of CCN protein to be replicated by small peptides that are not structurally constrained by disulfide bridges between cysteines, as in the complete domain III of CCN protein produced in eukaryotic systems. Furthermore, the inhibition of CCN2 activity by peptides derived from the primary sequence of domains I, III, and IV illustrates the lack of knowledge in the art as to whether peptides derived from specific domains of CCN2 can provide inhibition of the four-domain CCN protein.

[0016] Based on structure-activity analysis of CCN family proteins and the observation that CCN2 must undergo proteolytic processing to become biologically active, the inventors now find that the biologically active portion of the CCN5 protein is domain III, a thrombospondin type I repeat homology domain. This novel finding has led to the provision of biologically active structures based on domain III of CCN5 as well as domain III of other members of the CCN protein family. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] US2008 / 0207489 [Patent Document 2] EP2556839 [Non-patent literature]

[0018] [Non-licensed document 1] Perbal B,Tweedie SおよびBruford E,J Cell Commun Signal.2019 Sep;13(3):435 [Non-licensed document 2] Liuら,2017,Journal of Diabetes,9,pp.462-474 [Non-licensed document 3] Jun Lau,2011,Nat.Rev.Drug.Discovery,10(12),pp.945-963

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Non-licensed literature 9

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[0019] As a result of the insight into the structure-activity relationship of CCN5 and other CCN proteins, the present inventors provide novel biologically active recombinant proteins that reproduce the cell signaling and cell physiological functions due to CCN5 signaling and are also capable of counteracting other four-domain CCN proteins (Cyr61 (also known as CCN1), CTGF (also known as CCN2), NOV (also known as CCN3), WISP1 (also known as CCN4) and WISP3 (also known as CCN6). In other words, proteins are provided, including fusion protein type, based on domain III, the TSP1 homology domain of CCN protein, that reproduce or have the biological activity of CCN5 and are capable of antagonizing or inhibiting the effect of the four-domain CCN proteins, CCN1-4 or CCN6. In particular, the proteins herein have anti-fibrotic activity and may also have direct anti-cancer activity.

[0020] As mentioned above, it has been surprisingly found that domain III (TSP-1 homology domain) of other CCN proteins, i.e., four-domain CCN proteins, when provided as a separate domain in the absence of other CCN domains, is sufficient to reproduce the reported activity of CCN5. Thus, in other words, domain III of four-domain CCN proteins, when provided as a separate domain (i.e., as an isolated domain) in the absence of other CCN domains, has the same activity as CCN5, or in other words, domain III / TSP-1 homology domain of CCN5 (i.e., activity opposite to that of the full-length four-domain CCN protein). Thus, from the experiments disclosed in this document, it is clear that the isolated TSP-1 homology domain of any CCN protein can exert the same activity as that of the TSP-1 homology domain of CCN5. Other than in the case of CCN5, this may not be the same activity as that exerted by the full-length CCN protein.

[0021] Monomeric fusion proteins in which domain III of the CCN protein is fused to a monomeric fusion partner have been found to be particularly beneficial and useful in accordance with the present invention and the disclosures herein.

[0022] According to a first aspect, the present invention provides a monomeric fusion protein comprising: (i) a polypeptide corresponding to at least a portion of a thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein; (ii) a monomeric fusion partner fused at the N-terminus or C-terminus to the amino acid sequence of (i); and (iii) optionally, a peptide linker between the polypeptide of (i) and the monomeric fusion partner of (ii). Including, (i) the polypeptide is 40 to 60 amino acids in length and comprises an amino acid sequence selected from SEQ ID NO: 37 or 2 to 6, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 37 or 2 to 6, and all of the cysteine ​​residues in said sequence selected from SEQ ID NO: 37 or 2 to 6 are conserved; and (ii) the monomeric fusion partner and (iii) the peptide linker are not or do not contain an IGF-binding protein homology domain, a von Willebrand factor C-type repeat homology domain, or a cysteine ​​knot domain of a CCN family protein. The monomeric fusion protein is provided.

[0023] As will be described in more detail below, SEQ ID NOs: 37 and 2-6 correspond to 44 amino acid truncation fragments of domain III of CCN5, CCN3, CCN2, CCN1, CCN4 and CCN6, respectively, which contain the six conserved cysteine ​​residues of this domain. In particular, the fragments are flanked by the first and last cysteine ​​residues of the domain. Such fragments have been found to be particularly effective and resistant to proteolytic degradation.

[0024] In one embodiment, the polypeptide of (i) is: (a) an amino acid sequence selected from SEQ ID NO: 1 or 8-12; or (b) an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 or 8 to 12; or (c) a portion of the amino acid sequence of (a) or (b), comprising at least a 44 amino acid sequence of SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively. It comprises or consists of:

[0025] SEQ ID NOs: 1, and 8-12 correspond to slightly longer N-terminal truncated fragments of domain III of CCN5, CCN6, CCN3, CCN2, CCN1, and CCN4, respectively. These fragments contain the corresponding sequences of SEQ ID NOs: 37, 6, 2, 3, 4, and 5, respectively, along with some additional C-terminal sequence from the respective domain III.

[0026] In further embodiments, the polypeptide of (i) comprises an alanine residue at a position corresponding to position 2 of SEQ ID NO: 37, or 2-6, or said sequence selected from SEQ ID NO: 1, or 8-12. In some embodiments, the amino acid sequence of (i) comprises an amino acid sequence selected from SEQ ID NO: 38, or 42-46, or an amino acid sequence having at least 80% sequence identity thereto. In other embodiments, the amino acid sequence of (i) comprises an amino acid sequence selected from SEQ ID NO: 7, or 47-51, or an amino acid sequence having at least 80% sequence identity thereto. In this regard, substitution of this residue at position 2 has been found to be beneficial in promoting protein stability.

[0027] According to a further aspect of the invention, the monomeric fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110, 111, or an amino acid sequence having 80% sequence identity thereto.

[0028] In embodiments of these aspects, the monomeric fusion partner is selected from serum albumin, transferrin and a monomeric Fc fragment, particularly a monomeric Fc fragment of IgG, more particularly a monomeric Fc fragment of human IgG.

[0029] As mentioned above, the substitution at position 2 of the domain III fragment herein improves the stability of the fragment, in particular its resistance to protease degradation. Such sequence-modified variants of the domain III fragment herein represent proteins useful in themselves, without being linked to a fusion partner.

[0030] Thus, another aspect of the present invention also provides a protein of 40 to 60 amino acids in length comprising an amino acid sequence as set forth in SEQ ID NO: 7, 38, 42 to 46 or 47 to 51, or a sequence having at least 80% sequence identity thereto, or a protein consisting of such a sequence, which comprises an alanine residue at position corresponding to position 2 of said sequence in SEQ ID NO: 7, 38, 42 to 46, 47 to 51, and in which all of the cysteine ​​residues in said sequence are conserved.

[0031] As further aspects of the invention, other proteins and fusion proteins are also provided, as described in more detail below. According to a further aspect of the present invention there is provided a recombinant protein comprising formula (I): Cys-A-Cys-B-Cys-C-Cys-D-Cys-E-Cys-F (formula (I)) During the ceremony, A is of formula II A1-A2-A3-A4-A5-A6-A7-A8-A9 A peptide of the formula: wherein A1 is selected from the group consisting of P, A, V, I, and L; A2 is selected from the group consisting of E, D, A, I, L, and V; A3 is selected from the group consisting of G, Q, Y, S, N, W, F; A4 is selected from the group consisting of A, I, L, V, S, T; A5 is an amino acid selected from the group consisting of T, Y, N, G, Q, and S; A6 is an amino acid selected from the group consisting of A, V, I, L, P, S, E, D, K, R, and H; A7 is W; A8 is selected from the group consisting of G, T, S, Q, Y, N, P, A, V, I, and L; A9 is an amino acid selected from the group consisting of A, P, L, I, V, Q; and B is of formula III B1-B2-B3 wherein B1 is an amino acid selected from the group consisting of G, Q, N, S, Y, and T; B2 is an amino acid selected from the group consisting of T, S, N, F, Q, H, R, and K; B3 is an amino acid selected from the group consisting of G, Q, N, S, Y, and T; wherein one of B1-B3 is absent; and C is of formula IV C1-C2-C3-C4-C5-C6-C7-C8-C9-C10-C11-C12-C13-C14 wherein C1 is an amino acid selected from the group consisting of G, Q, N, S, Y, and T; C2 is an amino acid selected from the group consisting of K, R, H, M, T, S, A, L, I, and V; C3 is an amino acid selected from the group consisting of G, Q, N, S, Y, and T; C4 is an amino acid selected from the group consisting of M, F, A, I, L, V, and W; C5 is an amino acid selected from the group consisting of G, Q, N, S, T, Y, A, I, L, and V; C6 is an amino acid selected from the group consisting of G, Q, N, S, and T; C7 is an amino acid selected from the group consisting of H, R, and L; and C8 is an amino acid selected from the group consisting of A, L, I, and V. C9 is an amino acid selected from the group consisting of G, Q, N, S, T and Y; C10 is an amino acid selected from the group consisting of G, Q, N, S, T, Y (preferably N); C11 is an amino acid selected from the group consisting of V, P, A, I, L, G, Q, N, S, T, Y, R, K, D and E; C12 is an amino acid selected from the group consisting of G, Q, N, S, Y and T; C13 is an amino acid selected from the group consisting of H, K, R, A, L, I, V, P, G, Q, N, S, Y and T; C14 is an amino acid selected from the group consisting of F, P, W, G, Q, N, S, Y, T, E and D; and D is formula V D1-D2-D3-D4-D5-D6-D7-D8 D2 is an amino acid selected from the group consisting of P, A, L, I, V, M, W, D, and E; D3 is an amino acid selected from the group consisting of D, E, A, L, I, V, R, K, and H; D4 is an amino acid selected from the group consisting of G, Q, S, Y, T, R, L, K, and H; D5 is an amino acid selected from the group consisting of G, Q, N, S, Y, T, D, and E; D6 is an amino acid selected from the group consisting of H, R; K, G, Q, N, S, Y, and T; D7 is an amino acid selected from the group consisting of L, H, and R; D8 is an amino acid selected from the group consisting of A, L, I, and V; and E is of formula VI E1-E2-E3-E4 wherein E1 is an amino acid selected from the group consisting of P, A, L, I, V, M, W, G, Q, N, S, T, Y, D, and E; E2 is an amino acid selected from the group consisting of P, A, L, I, V, M, W, G, Q, N, S, T, Y; E3 is an amino acid selected from the group consisting of R, K, H, G, Q, N, S, T, and Y; E4 is an amino acid selected from the group consisting of P, A, L, I, and V; and F is absent or an amino acid sequence of up to about 13 amino acids. Contains 40 to 60 amino acids The recombinant protein is provided.

[0032] According to one embodiment of the above aspect, A1 is selected from the group consisting of P, I, and L; A2 is selected from the group consisting of E, V, and A; A3 is selected from the group consisting of W, Q, and Y; A4 is selected from the group consisting of S, T, and A; A5 is an amino acid selected from the group consisting of T and S; A6 is an amino acid selected from the group consisting of A, E, P, S, and K; A7 is W; A8 is selected from the group consisting of G, S, and T; A9 is an amino acid selected from the group consisting of P, Q, and A; and B1 is a serine (S); B2 is an amino acid selected from the group consisting of T, K, and R; B3 is an amino acid selected from the group consisting of T and S; and C1 is the amino acid G; C2 is an amino acid selected from the group consisting of T, L and M; C3 is G; C4 is an amino acid selected from the group consisting of M, F, I, and V; C5 is an amino acid selected from the group consisting of S and A; C6 is an amino acid selected from the group consisting of T and N; C7 is R; C8 is an amino acid selected from the group consisting of V, and I; C9 is an amino acid selected from the group consisting of S, and T; C10 is asparagine N; C11 is an amino acid selected from the group consisting of Q, R, D, V, and E; C12 is asparagine N; C13 is an amino acid selected from the group consisting of R, A, P, and S; C14 is an amino acid selected from the group consisting of F, Q, S, E, and N; and D1 is an amino acid selected from the group consisting of R, E, and W; D2 is an amino acid selected from the group consisting of L, M, and P; D3 is an amino acid selected from the group consisting of E, L, V, and R; D4 is an amino acid selected from the group consisting of T, K, and Q; D5 is an amino acid selected from the group consisting of Q and E; D6 is an amino acid selected from the group consisting of R, T, S, and K; D7 is arginine (R); D8 is an amino acid selected from the group consisting of L, and I; and E1 is an amino acid selected from the group consisting of L, M, E, N, and Y; E2 is an amino acid selected from the group consisting of S, V, L, and I; E3 is an amino acid selected from the group consisting of Q and R; E4 is P; F is absent or a peptide of up to 13 amino acids and comprises an amino acid sequence selected from the group consisting of PPSRGRSPQNSAF, GQPVYSSL, EADLEEN, EQEPEQPTD, DVDIHTLI, and DSNILKTIKIP, Contains a total of 44 to 57 amino acids A recombinant protein of formula (I) is provided.

[0033] According to a further embodiment of the above aspect, there is provided a recombinant protein of Formula I, wherein F is completely absent, partially absent, or is a peptide of about 13 amino acids comprising the amino acid sequence PPSRGRSPQNSAF.

[0034] More specifically, there is provided a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, SEQ ID NO:38; and fragments or variants thereof having greater than 50% sequence identity to the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, SEQ ID NO:38.

[0035] According to another aspect of the present invention there is provided a recombinant protein as defined above which is PEGylated. According to another aspect, the present invention provides (i) thrombospondin type 1 repeat (TSP-1) homology domains of CCN family proteins; (ii) a fusion partner fused N- or C-terminally to the TSP-1 repeat homology domain of (i) and selected from the group consisting of serum albumin, transferrin, and the Fc fragment of human IgG; (iii) optionally, a peptide linker between the TSP-1 repeat homology domain and the Fc fragment (fused at the N-terminus or C-terminus to the TSP-1 repeat homology domain of (i)). The present invention provides a fusion protein comprising:

[0036] According to an embodiment of the above aspect, there is provided a fusion protein comprising a recombinant protein according to the invention, as described above as a further aspect of the invention. The fusion partner of the fusion protein according to the invention is according to one embodiment selected from the group consisting of Fc fragments of IgG1, IgG2 or IgG4, serum albumin and transferrin.

[0037] According to a further embodiment, a fusion protein is provided, in which the fusion partner (ii) is an Fc fragment of IgG1, IgG2 or IgG4 containing a stabilizing disulfide bridge. Such a mutation may increase the thermal stability of the protein. Stabilizing mutations are known and have been reported in the art.

[0038] According to a still further embodiment, there is provided a fusion protein, wherein the fusion partner (ii) is an Fc fragment of IgG1, IgG2 or IgG4 comprising one or more mutations selected from the group consisting of S228P (for IgG4), E233P (for IgG1 and IgG4), F234A (for IgG4), L234A (for IgG1), L234V (for IgG1), F234V (for IgG4), L235A (for IgG1 and IgG4), ΔG236 (for IgG1 and IgG4) and ΔK447 (for IgG1, IgG2 and IgG4).

[0039] According to another embodiment, the fusion protein may comprise an Fc fragment selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0040] According to another embodiment, the fusion protein comprises a linker selected from the group consisting of SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:39.

[0041] According to one embodiment, the linker comprises the amino acid sequence (EAAAK)n, where n is at least 4, preferably n is 8. According to another embodiment, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27; SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:40 and SEQ ID NO:41.

[0042] According to another embodiment, the fusion partner in (ii) is serum albumin. According to a further embodiment of the second aspect of the invention, the fusion partner in (ii) is transferrin.

[0043] The present invention further provides according to a still further aspect a nucleic acid molecule (eg DNA) encoding a recombinant protein, protein or fusion protein according to the invention.

[0044] According to one embodiment of this aspect, there is provided a DNA sequence comprising a nucleic acid sequence as set forth in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113, and a nucleic acid sequence having about 80% sequence identity to SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113.

[0045] Furthermore, according to another aspect of the invention, there is provided an expression vector comprising a DNA sequence according to the invention. Also provided is a host cell comprising an expression vector according to the invention.

[0046] Finally, there are provided thrombospondin type 1 repeat (TSP-1) homology domains of CCN family proteins, proteins and fusion proteins as defined above for use as pharmaceuticals for treating or preventing disorders by inhibiting or counteracting cell signaling and cell physiological functions caused by four-domain CCN family proteins.

[0047] In one aspect there is provided a fusion protein or protein as defined herein for use in therapy. The fusion protein or protein may be for use in treating conditions associated with the activity of 4-domain CCN protein, particularly with undesirable or abnormal activity of 4-domain CCN protein. The activity may be associated with fibrotic effects. The activity may be profibrotic activity.

[0048] In another aspect, there is provided a fusion protein or protein as defined herein for use in the treatment or prevention of fibrosis or any condition exhibiting fibrosis (i.e. a fibrotic condition or disease). In a further aspect, there is provided a fusion protein or protein as defined herein for use in the treatment of cancer. There is also provided a fusion protein or protein as defined herein for use in the treatment of inflammatory or autoimmune diseases, or metabolic diseases.

[0049] According to this aspect of the invention there is also provided the use of a protein or fusion protein as defined herein for the manufacture of a medicament for treating or preventing a condition or disease as defined or described herein.

[0050] Such aspects also include compositions (e.g. pharmaceutical compositions) comprising a protein or fusion protein as defined herein for use in treating or preventing a condition or disease as defined or described herein.

[0051] These aspects also include a method of treating or preventing a condition or disease as defined or described herein, comprising the step of administering to a subject in need thereof an effective amount of a protein or fusion protein as defined herein, in particular said protein or fusion protein. [Brief description of the drawings]

[0052] [Figure 1-1] FIG. 1 shows the cell physiology and cell signaling of CCN5(dIII)-Fcv2 (one embodiment of the present invention, as defined in the sequence of SEQ ID NO: 28). A) shows that the CCN5(dIII)-Fcv2 fusion protein of SEQ ID NO: 28 causes concentration-dependent inhibition of Akt phosphorylation (serine-473) in A549 lung cancer cells. All error bars indicate SD. Statistical significance was calculated by one-way ANOVA with Dunnett's post-hoc test (p<0.05 is indicated by *). [Figure 1-2] FIG. 1 shows the cell physiology and cell signaling of CCN5(dIII)-Fcv2 (one embodiment of the present invention, as defined in sequence SEQ ID NO: 28). B) shows that the CCN5(dIII)-Fcv2 fusion protein of SEQ ID NO: 28 inhibits proliferation in a human lung fibroblast cell line, IMR90. All error bars indicate SD. Statistical significance was calculated by one-way ANOVA with Dunnett's post-hoc test (p<0.05 is indicated by *). [Figure 1-3] FIG. 1 shows the cell physiology and cell signaling of CCN5(dIII)-Fcv2 (one embodiment of the present invention, as defined in the sequence of SEQ ID NO: 28). C) shows that the CCN5(dIII)-Fcv2 fusion protein of SEQ ID NO: 28 inhibits the sphere-forming ability (anchoring-independent growth) of the estrogen receptor positive breast cancer cell line MCF-7 and the triple negative breast cancer cell line MDA-MB-231. All error bars indicate SD. Statistical significance was calculated by one-way ANOVA with Dunnett's post-hoc test (p<0.05 is indicated by *). [Figure 1-4]FIG. 1 shows the cell physiology and cell signaling of CCN5(dIII)-Fcv2 (one embodiment of the present invention, as defined in sequence SEQ ID NO:28). D) shows that CCN5(dIII)-Fcv2 fusion protein of SEQ ID NO:28 dose-dependently inhibits TGF-β-induced SMAD reporter activity (SMAD proteins are canonical TGF-β-regulated transcription factors). All error bars indicate SD. Statistical significance was calculated by one-way ANOVA with Dunnett's post-hoc test (p<0.05 is indicated by *). [Diagram 2] 2 shows the effect of different mutants of the hinge region of the Fc fragment on protease susceptibility of an embodiment of the invention in which CCN5(dIII) is fused to an Fc fragment of IgG, where the fusion proteins tested comprise sequences as shown in SEQ ID NO:28; SEQ ID NO:29, and SEQ ID NO:30, respectively. See Example 6 below. [Diagram 3] FIG. 3 shows the aggregation propensity of an embodiment of the invention depending on the structure of the peptide linker connecting the Fc fragment of IgG and CCN5(dIII), where the fusion proteins tested include sequences as shown in SEQ ID NO:30 and SEQ ID NO:31. [Figure 4] FIG. 4 illustrates a fusion protein according to the invention that comprises a TSP-1 repeat homology domain linked at its C-terminus to a peptide linker and to an Fc fragment through an Fc-hinge. [Diagram 5] FIG. 5 shows reduced susceptibility to endopeptidase cleavage when an embodiment of the invention incorporates a mutation of proline 195 in the CCN5 TSP-1 repeat homology domain, as shown in SEQ ID NO:7 (Fc-HLn8-CCN5(dIII)-P195A, SEQ ID NO:41), compared to the wild-type P195 variant of the CCN5 TSP-1 repeat homology domain (Fc-HLn8-CCN5(dIII), SEQ ID NO:40). [Figure 6]6 shows the production of a protein corresponding to SEQ ID NO:58, purified by Protein A capture chromatography. In the absence of the reducing agent, beta-mercaptoethanol (- lane), a dimer is seen to be present. However, in the presence of beta-mercaptoethanol (+ lane), the major product is seen to be a truncated fragment consisting of only the Fc fragment, rather than an intact fusion protein (TSP-1 homology domain fragment, peptide linker and Fc fragment) containing all of the portion encoded by SEQ ID NO:58. [Figure 7] 7 shows the production of a protein corresponding to SEQ ID NO:27 with a truncated C-terminal tail purified by Protein A capture chromatography, showing that the protein is significantly more resistant to protease degradation than the protein corresponding to SEQ ID NO:58, in which the C-terminal tail is included. [Figure 8] 8 shows the production of a protein corresponding to SEQ ID NO:73, similar to the protein corresponding to SEQ ID NO:27, purified by Protein A capture chromatography. Again, in the presence of beta-mercaptoethanol (+ lane), the protein is found to be more resistant to protease degradation than the protein corresponding to SEQ ID NO:58. [Figure 9] Figure 9 shows the results of an assay to measure phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of various concentrations of the protein corresponding to SEQ ID NO: 41 produced in stable transfected cells.It can be seen that the protein does not show any inhibition of the phosphorylation of AKT. [Figure 10] Figure 10 shows the results of an assay to measure phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of various concentrations of the protein corresponding to SEQ ID NO: 80 produced in stable transfected cells.It can be seen that the protein does not show any significant inhibition of the phosphorylation of AKT, and in fact, at higher concentrations, it can even cause an increase in phospho-AKT. [Figure 11]Figure 11 shows the results of an assay to measure phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of various concentrations of the protein corresponding to SEQ ID NO: 80 produced in transiently transfected cells. It can be seen that when produced in transiently transfected cells, the protein has concentration-dependent inhibitory activity against the phosphorylation of AKT. [Figure 12] 12 shows the results of an assay measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of various concentrations of proteins corresponding to SEQ ID NOs: 84, 94, and 106. It can be seen that each of these proteins has a concentration-dependent inhibitory activity against the phosphorylation of AKT. [Figure 13] Figure 13 shows the results of an assay measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of various concentrations of a protein corresponding to SEQ ID NO: 88. It can be seen that the protein is capable of inhibiting phosphorylation of AKT at concentrations above 10 μg / ml. [Figure 14] Figure 14 shows the results of an assay measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of various concentrations of proteins corresponding to SEQ ID NOs: 102 and 97. It can be seen that both proteins have concentration-dependent inhibitory activity against the phosphorylation of AKT. [Figure 15] 15 shows the results of an assay measuring phospho-AKT levels in A549 lung cancer cells after administration of various concentrations of a protein corresponding to SEQ ID NO: 110. It is found that the protein has a concentration-dependent inhibitory activity against the phosphorylation of AKT. [Figure 16-1] Figure 16 shows the results of several experiments involving the protein corresponding to SEQ ID NO: 106. A) It shows that the protein inhibits human lung fibroblast migration induced by both TGF-beta and CCN2. B) It shows that the protein inhibits scratch wound closure induced by both TGF-beta and CCN2. [Figure 16-2]Figure 16 shows the results of several experiments with the protein corresponding to SEQ ID NO: 106. C) It is shown that the protein produces partial inhibition of TGF-beta induction of expression of COL1A1, a gene known to be profibrotic. D) It is shown that the protein produces partial inhibition of TGF-beta induction of expression of FN1, a gene known to be profibrotic. [Figure 16-3] Figure 16 shows the results of several experiments with the protein corresponding to SEQ ID NO: 106. E) Shows that the protein produces partial inhibition of TGF-beta induction of expression of ACTA2, a gene known to be profibrotic. F) Shows that the protein produces partial inhibition of TGF-beta induction of expression of CCN2, a gene known to be profibrotic. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present invention is based on the surprising finding that the thrombospondin type 1 repeat (TSP-1) homology domain of CCN5, as mentioned, is a fully active structure that confers the cell signaling function of CCN5 / WISP2. Based on this novel insight into the activity of the TSP-1 repeat homology domain of CCN5, the inventors provide proteins, recombinant proteins and fusion proteins according to the present invention that can be used to inhibit or counteract the cell signaling and cell physiological functions due to the four-domain CCN proteins, namely CCN1, CCN2, CCN3, CCN4 and CCN6. Because it was not clear before which parts of full-length CCN5 are necessary to reproduce the activity of CCN5, and because full-length CCN molecules are highly susceptible to proteolysis and difficult to generate in an active homogenous form, this novel insight is considered to be of great importance to enable the formation of stable, homogenous drug-like molecules based on CCN5. Moreover, this implication that certain parts of CCN5 may be sufficient to reproduce the activity observed, for example, upon transient overexpression of the full-length protein, also contrasts with the general consensus in the art that CCN proteins act as matricellular proteins. The general consensus regarding the mechanism of action of CCN proteins, and matricellular proteins in general, is that, rather than acting as direct regulators of cell signaling, different segments of CCN proteins interact with a variety of other ECM proteins and cell surface receptors, thereby regulating their activity. The novel findings of the activity of the TSP-1 repeat homology domain of CCN5, and its structural close relationship with other members of the CCN protein family, suggest that the TSP-1 repeat homology domains of other CCN family proteins may also be utilized to inhibit the cell signaling function of four-domain CCN family proteins. According to one aspect, the recombinant and fusion proteins of the present invention inhibit the phosphorylation (Ser473) of AKT in A549 cells.

[0054] Inhibition of said cell signaling is suitable for the treatment of various disorders. For example, CCN2 is involved in several diseases, particularly those in which enhanced fibrogenesis and tissue fibrosis are characteristic pathophysiological features.

[0055] For example, overexpression of CCN2 alone has been shown to be sufficient to induce fibrosis in the lung (Sonnylal et al., Arthritis Rheum 62,1523-1532(2010)). CCN2 has also been found to be required for bleomycin-induced pulmonary fibrosis (Bonniaud P. et al. Am J Respir Cell Mol Biol 31,510-516(2004)), radiation-induced pulmonary fibrosis (Bickelhaupt,S. et al. J Natl Cancer Inst 109(2017)), and pulmonary fibrosis due to loss of PTEN (Phosphatase And Tensin Homolog) expression (Parapuram,SK et al. Matrix Biol 43,35-41(2015)). Furthermore, in the absence of other inducers, CCN2 has been found to induce pulmonary fibrosis when expressed in and secreted from pulmonary Clara cells (Wu, S. et al. Am J Respir Cell Mol Biol 42, 552-563 (2010)), alveolar type II epithelial cells (Chen, S. et al. Am J Physiol Lung Cell Mol Physiol 300, L330-340), when expressed from a fibroblast-specific promoter (Sonnylal et al. (2010) supra; Sonnylar, S. et al., J Cell Sci 126, 2164-2175 (2013)), or when delivered by adenovirus (Bonniaud, P. et al., Am J Respir Crit Care Med 168, 770-778 (2003)). Thus, numerous reports all support the conclusion that CCN2 is not only sufficient to induce fibrosis in the skin or lung, but is also required for a full fibrotic phenotype in several disease models. Pulmonary fibrosis is a hallmark of the human disease, idiopathic pulmonary fibrosis (IPF), but it also occurs in the setting of chronic obstructive pulmonary disease (COPD) (Jang, JH et al., COPD 14, 228-237 (2017)), and systemic sclerosis.Indeed, pulmonary fibrosis has been reported as the leading cause of death in up to 40% of patients with systemic sclerosis (Tyndall AJ et al., Ann Rheum Dis. 2010 Oct;69(10):1809-15). CCN2, and other CCN proteins such as WISP1, have also been implicated in the pathophysiology of both IPF (Konigshoff, M. et al., J Clin Invest 119,772-787(2009)) and COPD (Jang et al., supra) in human patients.

[0056] Another example is neoplastic disorders. For example, in the context of breast cancer, CCN2 has been shown to contribute to bone metastasis in a triple-negative breast cancer model (MDA-MB-231) (Kang, Y. et al., Cancer Cell 3, 537-549 (2003)). Furthermore, knocking down CCN2 in triple-negative breast cancer cells (MDA-MB-231), a cell line that expresses high levels of CCN2 (Chen, PS et al., J Cell Sci 120, 2053-2065 (2007)), reduced the migratory capacity of these cells, whereas overexpressing CCN2 in a hormone receptor-positive MCF-7 breast cancer cell line (Chen et al., supra), which has low endogenous expression of CCN2, increased the migratory capacity of the latter cells (Chen et al., supra; Chien, W. et al., Int J Oncol 38, 1741-1747 (2011)). A later report also found that overexpressing CCN2 in MCF-7 cells increased chemoresistance, whereas knocking down CCN2 in MDA-MB-231 cells decreased chemoresistance (Wang, MY, et al., Cancer Res 69, 3482-3491 (2009)). Increased chemoresistance conferred by CCN2 has also been reported for other breast cancer cells (Lai, D, et al., Cancer Res 71, 2728-2738 (2011)). Furthermore, through overexpression or knockdown studies, CCN2 has also been shown to contribute to epithelial to mesenchymal transition (EMT) and increased anchorage-independent growth (mammosphere formation) capacity of breast cancer cells (Chen et al., supra; Zhu, X., et al., Oncotarget 6, 25320-25338 (2015)). Both findings of increased chemoresistance and enhanced EMT induced by CCN2 are consistent with the association between EMT and chemoresistance in other cancer types as well (Fischer, KR et al., Nature 527, 472-476 (2015); Zheng, X. et al., Nature 527, 525-530 (2015)).

[0057] In a particular aspect, the present invention provides a monomeric fusion protein as defined above, comprising a polypeptide corresponding to at least a portion of a thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein, wherein the TSP-1 homology domain sequence may be truncated and / or modified, but the cysteine ​​residues of the domain are conserved. This polypeptide may be conveniently referred to as a "TSP-1 polypeptide", and this term is therefore to be understood as not having or indicating any limitation to only a specific native TSP-1 homology domain sequence. The term "TSP-1 polypeptide" may be used synonymously or interchangeably with "TSP-1 domain protein" or "TSP-1 domain sequence".

[0058] As demonstrated in the Examples below, it has surprisingly been found that monomeric fusion partners are preferred in producing active and stable proteins as compared to dimeric fusion partners, such as Fc fragments from IgG proteins, which result in dimeric fusion proteins. Monomeric fusions retain the activity of the TSP-1 domain polypeptides they contain. Furthermore, the proteins are stable, including with respect to proteolytic degradation. As further described below, resistance to proteolytic degradation can be improved by making modifications to the amino acid sequence of the TSP-1 polypeptide, including, inter alia, the Ala substitutions discussed above.

[0059] Thus, the polypeptide of component (i) of the fusion protein retains at least 80% sequence identity to said sequence selected from SEQ ID NO: 37 or 2-6, or to a sequence selected from SEQ ID NO: 1 or 8-12, and may contain insertions, deletions, substitutions, mutations or any combination thereof relative to said sequence, so long as all of the cysteine ​​residues in said sequence are conserved.

[0060] In another aspect, the invention provides a protein (e.g., a recombinant protein) consisting of or comprising a polypeptide corresponding to at least a portion of a thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein, but not in the context of a fusion protein, where the TSP-1 domain sequence may be truncated and / or modified and includes an Ala substitution at a position corresponding to SEQ ID NO: 37 or 2-6, or position 2 of SEQ ID NO: 1 or 8-12, but the cysteine ​​residues of the domain are conserved. In other words, the TSP-1 domain protein may be provided without or independent of another component, such as a fusion partner. Thus, the TSP-1 domain protein is not fused or linked to another protein domain or component, or to other functional or structural protein sequences. For convenience, such proteins may be referred to as "Ala-substituted proteins".

[0061] As used herein, the term "conserved" means that no residues in a given sequence have been deleted or substituted. In other words, the term "conserved" is used synonymously (and interchangeably) with the term "retained". This simply means that no cysteine ​​residues have been removed from the sequence. Thus, in the above context, this means that no cysteine ​​residues in the sequence selected from SEQ ID NO: 37 or 2-6 or 1 or 8-12 have been deleted or substituted. It is noted that the insertion of additional residues between the conserved residues (e.g., between the conserved cysteines) or the deletion of non-conserved (e.g., non-cysteine) residues may alter the position of the conserved residue in the polypeptide sequence relative to its position in the original reference sequence (e.g., a sequence selected from SEQ ID NO: 37 or 2-6). However, such residues are still considered "conserved" as defined herein. Thus, the term "conserved" does not impose any restriction or limitation on the position (or more specifically, the position number) of the cysteine ​​residue.

[0062] In some embodiments, the polypeptide of (i) is: (a) an amino acid sequence selected from SEQ ID NO: 1 or 8-12; or (b) an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 or 8 to 12; or (c) a portion of the amino acid sequence of (a) or (b), comprising at least a 44 amino acid sequence of SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively. It comprises or consists of:

[0063] As mentioned above, the (ii) monomeric fusion partner and (iii) peptide linker are not or do not contain the IGF binding protein homology domain, von Willebrand factor type C repeat homology domain, or cysteine ​​knot domain of the CCN family protein. In other words, the only domain of the CCN family protein that may be present in the fusion protein of the invention is the TSP-1 homology domain.

[0064] Likewise and similarly, in the context of an Ala-substituted protein that is not a fusion protein, the protein does not contain any other CCN domains (other than the TSP-1 domain protein).

[0065] In some embodiments, the polypeptide or Ala-substituted protein of (i) may only comprise a portion of the TSP-1 homology domain, as defined above. The inventors have found that the minimal fragment of the TSP-1 domain required is the 44 amino acid sequence of SEQ ID NO: 37, 6, 2, 3, 4 or 5. Thus, in some embodiments, the polypeptide of (i) is at least 44 amino acids in length. In some embodiments, the polypeptide of (i) is 44-57 amino acids in length. However, as described above, there may be one or more deletions of amino acids in the 44 amino acid minimal fragment located between the cysteine ​​residues. Thus, in some embodiments, the length of the TSP-1 polypeptide may be less than 44 residues, i.e., 40-43 residues.

[0066] In some embodiments, the polypeptide of (i) consists of an amino acid sequence selected from SEQ ID NO: 37 or 2-6, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 37 or 2-6.

[0067] As mentioned above, the protein of the present invention, including fusion protein, exhibits (or in other words, exhibits or has) the activity, more specifically biological activity, of CCN5. In one embodiment, the protein may retain or exhibit or have the activity of the TSP-1 homology domain of CCN5. Alternatively, the protein may be defined as exhibiting (or exhibiting or having) the activity, particularly biological activity, of the isolated TSP-1 homology domain of CCN protein. The foregoing may apply to any activity of the domain, and in particular to the activity reflecting the anti-fibrotic effect of the TSP-1 homology domain. Based on any particular biological effect of the domain, any suitable assay or method may be used to assay for (or test or detect) such activity.

[0068] It is noted that the activity of a given protein can be suitably evaluated by analyzing the effect of the protein on the phosphorylation of AKT.In particular, as described in Example 2, a given protein can be assayed for its ability to inhibit the phosphorylation (Ser-473) of AKT in A549 human lung cancer cells.Those skilled in the art will recognize that other similar assays can be devised to evaluate the same activity or other related antifibrotic activity.

[0069] As mentioned above, in another aspect of the present invention, there is provided a recombinant protein that inhibits or counteracts cell signaling and cell physiological functions caused by a four-domain CCN protein, said recombinant protein comprising an amino acid sequence as set forth in formula I above.

[0070] Cys-A-Cys-B-Cys-C-Cys-D-Cys-E-Cys-F where A, B, C, D, E and F are as defined above and in the accompanying claims. Formula I is the result of an alignment of the TSP-1 repeat homology domains of structurally related CCN family proteins (CCN1-CCN6), which all contain six conserved cysteines, and takes into account that amino acids may be substituted without affecting the activity of the protein (conservative substitutions, as discussed further below). The position of the first conserved cysteine ​​of the TSP1 repeat homology domains of the different CCN proteins is defined as position 1 of the recombinant protein of Formula I.

[0071] The five segments between the conserved cysteines are A, B, C, D, and E, respectively. The first segment A is defined by the formula A1-A2-A3-A4-A5-A6-A7-A8-A9, where A1 through A9 are as defined above. The amino acid in the seventh position (A7) of segment A is tryptophan (W) in all members of the CNN family of proteins and is considered to be conserved.

[0072] The second segment B is defined by the formula B1-B2-B3, where B1 to B3 are as defined above. According to one embodiment, B1 and B3 are either serine or threonine.

[0073] The third segment C has the formula C1-C2-C3-C4-C5-C6-C7-C8-C9-C10-C11-C12-C13-C14, where the amino acids C1 to C14 are as defined above. According to one embodiment, the amino acids C1 and C3 are glycine (G). According to another embodiment, C7 is arginine (R) and C10 and C12 are both asparagine (N).

[0074] The fourth segment D is defined by the formula D1-D2-D3-D4-D5-D6-D7-D8, where the amino acids D1 to D8 are as defined above. According to one embodiment, D7 is arginine (R).

[0075] The fifth segment E is defined by the formula E1-E2-E3-E4, where the amino acids E1 to E4 are as defined above. According to one embodiment of the invention, E4 is a proline.

[0076] The final cysteine ​​is followed by a carboxyl-terminal peptide segment (F) of variable length, containing 0 to 13 amino acids. F may be deleted or truncated compared to the amino acid sequence of the TSP-1 repeat homology domain of the CCN family protein. According to one embodiment, F is absent. According to another embodiment, F consists of a peptide selected from the group consisting of PPSRGRSPQNSAF, GQPVYSSL, EADLEEN, EQEPEQPTD, DVDIHTLI, and DSNILKTIKIP. According to one aspect of this embodiment, the recombinant protein may take the form of an amino acid sequence as shown in SEQ ID NOs: 8-12.

[0077] According to another aspect, the present invention provides a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, SEQ ID NO:38; and fragments or variants thereof having at least 50% sequence identity to the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, SEQ ID NO:38.

[0078] According to one aspect, there is provided a recombinant protein consisting of amino acids selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, SEQ ID NO:38; and fragments or variants thereof having greater than 50% sequence identity to the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, SEQ ID NO:38.

[0079] A "recombinant protein," as used herein, is a protein encoded by a recombinant nucleic acid. These are expressed from recombinant nucleic acids in a host cell, as further disclosed below.

[0080] "Recombinant nucleic acid," as used herein, describes a nucleic acid molecule that, by its origin or manipulation, is not associated with all or a portion of a polynucleotide with which it is naturally associated, and / or is linked to a polynucleotide other than that with which it is naturally linked, as further disclosed below.

[0081] Those skilled in the art know that modifications in the amino acid sequences of recombinant and fusion proteins according to the invention can be introduced without altering the activity of said proteins. Amino acids are usually classified as hydrophobic or hydrophilic and / or as having polar or non-polar side chains. The replacement of one amino acid with another that has the same biochemical properties is generally known as a conservative substitution.

[0082] Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: ·MILV FYW ·KRH ·AG ·ST QN ED In general, conservative amino acid substitutions refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made, and therefore do not alter the structure of the protein significantly, which is why biological activity is also not significantly altered.

[0083] Those skilled in the art know that the biological activity of a protein can also be retained even if one or several amino acids are deleted, inserted or added to the amino acid sequence, so long as the structural and chemical-physical properties are preserved.

[0084] The symbol "Δ" when used immediately before an amino acid refers to a deletion of the indicated amino acid, for example, ΔK447 shall be understood as the protein without K447. Deletions of specific amino acids are also indicated herein with a "-" instead, for example, K447- shall also be understood as the protein without K447.

[0085] Thus, the present invention includes recombinant and fusion proteins as disclosed in the appended claims, and it is to be understood that such modifications (amino acid substitutions, deletions, insertions and additions) as described above may be introduced without essentially altering their biological activity, i.e., their ability to inhibit or counteract cell signaling and cell physiological functions attributable to the four domain CCN family proteins; CCN1, CCN2, CCN3, CCN4 and CCN6.

[0086] Throughout this specification, reference is made to amino acid sequences. When referring to amino acid sequences herein, sometimes the present specification refers to modifications of the amino acid sequence or protein in question by referring to "uniprot numbering" or Eu numbering. Uniprot numbering refers to the numbering used in the uniprot database (UniProt Consortium, Nucleic Acids Res. 2019 Jan 8;47(D1):D506-D515). When referring to amino acid numbers of CCN proteins, uniprot numbering is used. Eu numbering refers to the numbering of Eu antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85) and is used when referring to amino acids in the Fc fragment of human IgG subclasses with or without mutations or chimeras that differ from the wild type. The Eu numbering system is accessible, for example, from the International ImMunoGeneTics Information System (IMGT) in the IMGT Scientific chart. IMGT is described in Lefranc MP, Biomolecules. 2014 Dec;4(4):1102-1139.

[0087] As used herein, when referring to "sequence identity," a sequence having at least x% identity to a second sequence refers to the number of amino acids in the first sequence that are identical to matching amino acids in the second sequence when both sequences are optimally aligned through a global alignment over the entire length of the second amino acid sequence. When x is at its maximum, both sequences are optimally aligned. Alignment and percent identity determination may be performed manually or automatically.

[0088] Alignment for purposes of determining percent amino acid sequence identity may be accomplished in a variety of ways within the skill of the art, for example using publicly available software such as ClustalOmega (Sievers F, Higgins DG (2018) Protein Sci 27:135-145), Protein BLAST (National Center for Biotechnology Information (NCBI), USA), or commercially available software such as Megalign (DNASTAR) software. Those skilled in the art may determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. NCBI BLAST is another example of software used to determine amino acid sequence identity (MacWilliam et al., Nucleic Acids Res. 2013 Jul;41 (Web Server issue):W597-W600).

[0089] According to one aspect of the present invention, there is provided a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, and SEQ ID NO:38; and fragments or variants thereof having at least 50% sequence identity to the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, and SEQ ID NO:38.

[0090] According to another aspect, there is provided a recombinant protein comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, and SEQ ID NO:38.

[0091] Biologically active proteins and peptides have an important place in the clinical management of human diseases. However, many proteins and peptides present difficulties because they are removed by renal filtration due to their small size and / or because of proteolytic metabolism, and therefore have less than ideal pharmacokinetic properties. These factors may also impose limitations or difficulties when administering drugs to subjects in need of treatment, such as the need to administer constant infusions or frequent subcutaneous administrations to maintain the circulating concentration of the protein or peptide at an effective therapeutic level. The need for constant or very frequent administration of drugs is clinically undesirable, as it presents obvious difficulties and inconveniences to both patients and physicians.

[0092] One strategy for extending the half-life of biologically active peptides or proteins is to link polyethylene glycol (PEG) groups to the peptide or protein of interest by a process called PEGylation (see, for example, Dozie et al. (2015), Int. J. Mol Sci, 16(10) 25831-25864). A common strategy for protein PEGylation is to react a functional group on the protein with a complementary group on a PEG molecule to form a protein-PEG conjugate. The PEG moiety offers several advantages for increasing protein stability and circulating half-life due to its flexibility, hydrophilicity, diverse size and low toxicity.

[0093] In one embodiment, the present invention therefore provides a recombinant protein as described above, wherein said protein is PEGylated. The fusion protein according to the present invention may also be PEGylated.

[0094] Fusion proteins Another way to eliminate the difficulties with the medical use of peptides and proteins is to obtain an extension of the half-life of a biologically active protein or peptide by creating a fusion protein (see, for example, Valeria et al. (2017), “A New Approach to Drug Therapy: Fc-Fusion Technology”, Prim Health Care, 7:255, doi:10.4172 / 2167-1079.1000255). Through genetic engineering, it is possible to increase the molecular weight of the protein of interest to approximately 60-70 kDa, which is the threshold for renal filtration, by covalently fusing the protein or peptide to a carrier protein.

[0095] The present invention relates to (i) thrombospondin type 1 repeat (TSP-1) homology domains of CCN family proteins; (ii) a fusion partner fused N- or C-terminally to the TSP-1 repeat homology domain of (i) and selected from the group consisting of serum albumin, transferrin, and an immunoglobulin Fc fragment; (iii) optionally, a peptide linker between the TSP-1 repeat homology domain of (i) and the Fc fragment (fused at the N- or C-terminus to the TSP-1 repeat homology domain). The present invention provides a fusion protein comprising:

[0096] Throughout this specification, the TSP-1 repeat homology domain may also be referred to as and referred to as domain III, which refers to the third domain of the CCN family proteins.

[0097] In a preferred aspect, the fusion partner is a monomeric fusion partner and results in a fusion protein that is monomeric. Such a fusion protein, and in particular the TSP-1 domain thereof, is defined above and further described below.

[0098] However, the disclosure also includes other embodiments with respect to both the TSP-1 domain protein component and the fusion partner component. According to such an embodiment, the TSP-1 repeat homology domain is a recombinant protein of Formula I, as defined above.

[0099] According to another embodiment, the TSP-1 repeat homology domain is a recombinant protein having an amino acid sequence as defined in any one of the sequences shown in SEQ ID NOs: 1-12, 37, and 38, or a recombinant protein of formula I as defined above.

[0100] According to one embodiment, the TSP-1 repeat homology domain is a recombinant protein comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, and SEQ ID NO:38.

[0101] Proteins are inherently susceptible to protease degradation. To prevent protease degradation of recombinant and fusion proteins according to the invention, modifications to the amino acid sequence may be introduced, for example by site-directed mutagenesis, to provide protease-resistant recombinant and fusion proteins. For example, a point mutation may be introduced in the thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein as defined in SEQ ID NO: 1-12, 37 or 38, or more specifically of a protein as defined in any one of SEQ ID NO: 1-6, 8-12 or 37, of the fusion protein or in the recombinant protein. According to one embodiment, a point mutation is introduced that reduces the susceptibility to proteolytic degradation. A non-limiting example of a point mutation that results in less proteolysis of the recombinant and fusion proteins of the invention is by introducing a point mutation corresponding to the substitution of proline at position 195 in domain III of CCN5, e.g. as shown in SEQ ID NO: 7, with alanine (P195A). Similar mutations may also be introduced in the amino acid sequence resulting from domain III of other CCN family members. SEQ ID NO: 38 corresponds to a truncated 44 amino acid sequence of the TSP-1 domain of CCN5, containing an Ala substitution. SEQ ID NOs: 42-46 correspond to truncated 44 amino acid sequences of the TSP-1 homologous domain of CCN1, 2, 3, 4 and 6, respectively, containing an Ala substitution. SEQ ID NOs: 47-51 correspond to longer TSP-1 homologous sequences of CCN1, 2, 3, 4 and 6, respectively, containing an Ala substitution. Any such sequence, or sequences having at least 80% sequence identity thereto, may be used in accordance with the present invention.

[0102] As mentioned above, in a preferred embodiment, the fusion partner (ii) of the fusion protein according to the invention is monomeric. Any monomeric fusion partner may be used. Thus, the fusion partner may be any protein, or portion thereof (e.g., a protein domain), that exists and remains in monomeric form when fused to the TSP-1 homology domain protein component. Thus, the fusion protein comprising a monomeric fusion partner and a TSP-1 homology domain protein remains monomeric, i.e., does not dimerize or form higher order multimers with itself.

[0103] A wide variety of proteins are known as possible fusion partners and may include naturally occurring proteins, or fragments or amino acid sequence modified variants thereof, as well as synthetic proteins or amino acid homopolymers, including the Fc fragment of IgG, serum albumin or transferrin, among others.

[0104] A fusion partner is broadly defined herein as a second polypeptide (or second amino acid sequence) that is not naturally associated (e.g., adjacent or directly or indirectly linked) with a first CCN TSP-1 homologous polypeptide and is linked to the first CCN TSP-1 homologous polypeptide in a synthetic or artificial combination. Thus, a fusion protein includes a non-natural combination of at least two amino acid sequences or polypeptides that are linked or fused together.

[0105] The fusion partner may be an amino acid sequence of at least 6, 8, 9, 10, 15, 20, 25, 30, 40 or 50 or more amino acids in length. Typically, the fusion partner is a functional polypeptide, or in other words, a polypeptide that confers a function or property to the fusion protein, such as stabilizing the fusion protein (making the first polypeptide more stable) or increasing serum half-life. Thus, the fusion partner may be a structural protein or have a structural function, or the fusion partner may confer an activity or property to the fusion protein, such as binding activity (e.g., the fusion partner may be a member of a binding pair or may be an affinity binding partner, etc.). In representative examples, the fusion partner may be albumin (particularly serum albumin), fibrinogen, glutathione S-transferase, transferrin, streptavidin or streptavidin-like protein, or an immunoglobulin, or a portion thereof, in particular the Fc portion of an immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4), or a portion or modification thereof. Suitable serum albumins include bovine serum albumin (BSA), mouse serum albumin (MSA) and especially human serum albumin (HSA). Other possible fusion partners include polypeptides that can act to improve the pharmacokinetic properties of the fusion protein, such as synthetic polypeptides, such as homoamino acid polymers, proline-alanine-serine polymers, or elastin-like peptides, such as those described in Strohl, 2015, BioDrugs 29, 215-239. Any fusion partner known in the art for use with therapeutic proteins may be used.

[0106] In one embodiment, the fusion partner (ii) of the fusion protein according to the invention may be either the Fc fragment of IgG (any subclass or chimera of any subclass), serum albumin or transferrin.

[0107] The fusion partner may be N- or C-terminally coupled to the TSP-1 homology domain protein component of the fusion protein, e.g., to the TSP-1 repeat homology domain of either CCN5 or another CCN protein, as defined herein, or may be linked directly or indirectly through a linker, as further described below.

[0108] Fc fragments tend to form dimers, and when used in fusion proteins, the fusion protein construct tends to contain two copies of the fusion protein, however, it is known in the art that monomeric Fc fragments and monomeric fusion proteins containing them can be prepared.

[0109] Thus, when the fusion partner is an Fc fragment, it is preferably a monomeric Fc fragment, such as a monomeric Fc fragment of any class of human IgG. Chimeric Fc fragments containing portions of Fc regions from different classes are included, as are Fc fragments containing modified sequences.

[0110] Fc fusion proteins are a growing field of protein therapeutics based on chimeric proteins consisting of an effector domain coupled to an Fc fragment of an IgG isotype. A typical example of a biopharmaceutical product is etanercept (TNF-α receptor coupled to an Fc fragment) used to treat rheumatoid arthritis. Another example of an Fc fusion biopharmaceutical protein is aflibercept, a VEGF receptor-Fc fusion protein used to treat exudative macular degeneration and metastatic colorectal cancer. The main rationale for creating Fc fragment fusion proteins is to obtain an extended half-life due to a sufficient increase in molecular weight to eliminate renal excretion and to enhance renal proximal tubule reabsorption through neonatal Fc receptors. Also, the pH-dependent binding of Fc fusion proteins to neonatal Fc receptors (FcRn) on endothelial cells allows Fc-based fusion proteins that would otherwise be destined for endocytosis and subsequent lysosomal degradation to be recycled and released back into the circulation.

[0111] According to one embodiment, a fusion protein is provided in which the fusion partner (ii) is an Fc fragment from human IgG (immunoglobulin G, also known as immunoglobulin gamma), including all subclasses of human IgG. According to yet another embodiment of the invention, a fusion protein is provided in which the fusion partner is an Fc fragment of IgG1, IgG2 or IgG4. Preferably, the Fc fragment of human IgG is of subclass IgG4 (SEQ ID NO: 13) or IgG2 (SEQ ID NO: 14).

[0112] IgG1, IgG2 and IgG4 are often preferred over IgG3 because they have a longer half-life of approximately 3 weeks. Those skilled in the art will know that the selection of a particular subclass of IgG isotype as an Fc fusion partner depends on the desired half-life extension and cytotoxicity level of activity of the final compound. Therapeutic antibodies indicated for the treatment of cancer or autoimmune diseases belong to the IgG1 subclass in most cases because of their high affinity to Fc receptors and their strong ability to exert immune effector functions. On the other hand, IgG2 and IgG4 are preferred IgG subclasses for use as the backbone of therapeutic candidates when the lack of immune effector functions is desired because immune effector functions may cause adverse effects. The tendency of Fc fragments to activate immune effector functions depends on the Ig isotype and subclass, and is diverse for different immune effectors. In addition to selecting the appropriate IgG subclass Fc fragment, the amino acid sequence of the IgG subclass may be modified, for example by site-directed mutagenesis, to reduce the ability of the Fc fragment to activate immune effector functions.

[0113] One may select Fc fragments that form monomers, or more precisely, that retain or have a monomeric form, or the Fc fragment may be modified to introduce mutations that allow or promote a monomeric structure. Such mutations are referred to herein as "monomer-generating mutations". Examples of Fc fragments that contain monomer-generating mutations are SEQ ID NO: 54 and SEQ ID NO: 55. Those skilled in the art know how to introduce such mutations and select monomeric Fc mutants.

[0114] Evasion of immune effector activation functions of Fc fragments For example, the biologic fusion protein dulaglutide (Trulicity) is a GLP-1 agonist-Fc fragment fusion protein for the once-weekly treatment of type 2 diabetes. TM ), the well-characterized mutations F234A and L235A have been introduced into the hinge region of an IgG4 Fc fragment to reduce the capacity for activated immune effector functions.

[0115] According to one embodiment of the invention, the Fc fusion partner is an Fc fragment of IgG4, where the IgG4 Fc fragment has been modified to avoid immune effector function, for example to include the F234A and L235A mutations described above.

[0116] Protease-resistant Fc fragment Another factor that can reduce both the yield during production and the biological half-life is the endopeptidase cleavage of fusion proteins. To reduce or eliminate the risk of proteolytic degradation, modifications may be introduced into the amino acid sequence of the Fc fragment, particularly by introducing mutations at sites that are sensitive to proteolytic cleavage. In EP patent application EP2654780B1, the Fc domain of the IgG1 constant region was modified by replacing E233-L234-L235-G236 with P233-V234-A235 (deleting G236) (EU numbering), making the resulting modified Fc-containing protein resistant to proteolytic degradation.

[0117] Incorporation of the amino acid modifications disclosed in EP2654780B1 in an IgG4 Fc fragment coupled to domain III of CCN5 was considered not to provide sufficient resistance to endopeptidases, however, improved protease resistance was achieved by further modification of the IgG subtype used as fusion partner according to the present invention.

[0118] More specifically, fusion proteins containing the entire hinge region of IgG2 and the constant heavy chains 2 and 3 of IgG4 have been found to exhibit excellent resistance to proteolysis. Mueller JP et al., Mol. Immunol. (1997), 34(6), pp. 441-452, disclose the use of IgG2 / IG4 chimeras in IgG antibodies. Another biologic monoclonal antibody called eculizumab, used to treat paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome, has been shown to be useful, for example, in circumventing the ability of IgG4 to activate FcγR-dependent immune effectors. Furthermore, it has also been shown that the IgG4 constant domains 2 and 3 of such chimeric Fc fragments circumvent the ability of IgG2 to activate complement-dependent immune effector functions. In this regard, see the report by Rother et al. (2007), cf. Nat. Biotechnol., 25(11), pp. 1256-1264 and Mueller JP et al., supra.

[0119] Also, Borrok et al. (2017), J. Pharm. Sci.106;1008-1017 disclose the introduction of modifications in the Fc fragment to study their impact on the immune effector function of the antibody (FQQ-YTE mutations). In WO2017158426A1, modifications of antibodies by introducing mutations in the Fc fragment to improve the half-life of the antibody are disclosed. In particular, modifications of one or more of positions 311, 434, 428, 438, and 435 in the Fc region of the immunoglobulin are disclosed.

[0120] Furthermore, Kinder et al. J Biol Chem. 2013 Oct 25;288(43):30843-54 report that mutations in the lower hinge of IgG1 (i.e., E233P, L234V, L235A, G236-, Eu numbering) resulted in a protease-resistant IgG1 antibody.

[0121] According to one embodiment, the Fc fragment of the fusion protein according to the invention consists of an Fc fragment of IgG4 subclass incorporating the following mutations; S228P, F234A, L235A, K447-, Eu numbering, see SEQ ID NO: 15.

[0122] Jacobsen et al. J Biol Chem. 2017 Feb 3;292(5):1865-1875 reported mutations of Asn297 that result in aglycosylation of the Fc fragment and further result in the loss of IgG effector function. Jacobsen also found that some mutants (N297G) result in antibodies with better stability and developability compared to other mutants (N297Q or N297A). Further modifications that result in better stability than the parent IgG1 were also introduced (disulfide bridges).

[0123] According to the invention, when the fusion partner is an Fc fragment, this may not contain or may contain stabilizing disulfide bridges, as in SEQ ID NO:16, and may be aglycosylated.

[0124] To the best of our knowledge, the Fc fragment, which is composed of the entire hinge region of IgG2 and the constant heavy chains 2 and 3 of IgG4, has not been used to prepare a fusion protein by linking said Fc fragment to an effector protein.

[0125] According to one embodiment, the fusion partner of this fusion protein is an Fc fragment of IgG1, aglycosylated and stabilized by a disulfide bridge and in which a lower hinge containing the following mutations: E233P, L234V, L235A, G236- (Eu numbering) has been introduced (SEQ ID NO: 17).

[0126] According to one embodiment, the fusion partner of the fusion protein comprising the TSP-1 repeat homology domain of a CCN family protein is the Fc fragment of IgG4, and in addition to the S228P and K477-mutations, the following mutations are introduced in the lower hinge: E233P, L234V, L235A, G236-(Eu numbering) (SEQ ID NO: 18).

[0127] In one preferred embodiment, the Fc fragment is a chimera of the hinge region of IgG2 (216 ERKCCVECPPCPAPPVA-GP 238, Eu numbering) and any other IgG subclass. Most preferably, the Fc fragment is a chimera of the constant heavy domains 2 and 3 of IgG4 with a deletion of the hinge region of IgG2 and the carboxyl terminal K477 (Eu numbering) as shown in SEQ ID NO: 19. This embodiment of the invention has been shown to have improved protease resistance properties (see Example 6).

[0128] In one embodiment, the fusion partner of the monomeric fusion protein of the invention is an IgG1 Fc fragment that is disulfide bridge stabilized (R292C, V302C), aglycosylated (N297G) and with monomer generating mutations (C220Q, C226Q, C229Q, T366R, L368H, P395K, K409T, M428L, Eu numbering) as provided in SEQ ID NO:54.

[0129] In a further embodiment, the fusion partner of the monomeric fusion protein of the invention is an Fc fragment that is a chimera of the hinge region of IgG2 and the constant heavy chain domains 2 and 3 of IgG4, including the carboxyl terminal K477- deletion and monomer generating mutations (C219Q, C220Q, C226Q, C229Q, L351F, T366R, P395K, F405R, Y407E) and half-life extending mutations (M252Y, S254T, T256E) (Eu numbering), as provided in SEQ ID NO:55.

[0130] The fusion proteins according to the invention are exemplified by using an Fc fragment consisting of the entire hinge region of IgG2, constant heavy chains 2 and 3 of IgG4, and domain III of a CCN protein family member, although it is believed that suitable protease resistance will also be achieved by coupling such Fc fragment chimeras to other effector molecules, such as VEGFR, FGF-21 or GLP1. The effector molecule is the part of the Fc fusion protein that confers the desired pharmacokinetic properties, while the Fc fragment contributes to the pharmacodynamic properties.

[0131] Serum albumin as a fusion partner Another strategy to extend the half-life of peptides and proteins is to use serum albumin (SA) as a fusion partner. IgG and SA share an extended half-life of about 19 days, compared to a few days or less for most other circulating proteins. SA also has affinity for the nascent Fc receptor (FcRn) and is rescued from intracellular degradation (see Andersen et al. (2014), J Biol Chem, 289(19); pp13492-13502).

[0132] In one embodiment of the invention there is provided a fusion protein as described above, wherein the fusion partner is serum albumin, preferably human serum albumin. In one embodiment, a monomeric fusion protein as described above is provided, wherein the fusion protein comprises amino acids 25-606 of human serum albumin, as provided in SEQ ID NO:101.

[0133] In further embodiments of the invention, albumin, e.g., human serum albumin, has been modified to increase or decrease its half-life, e.g., by altering its FcRn affinity, with or without pH dependency, resulting in increased or decreased half-life.

[0134] Transferrin as a fusion partner Yet another strategy for extending the half-life of peptides and proteins is to use transferrin as a fusion partner, taking advantage of the naturally long half-life of transferrin (Strohl W. BioDrugs. 2015;29(4):215-239). Either glycosylated or non-glycosylated forms of transferrin may be used.

[0135] In one embodiment of the invention there is provided a fusion protein as described above, wherein the fusion partner is transferrin, preferably human transferrin. In one embodiment, a monomeric fusion protein as described above is provided, wherein the fusion protein comprises amino acids 20-698 of human transferrin, as provided in SEQ ID NO:53.

[0136] Linker According to another embodiment, the fusion protein according to the invention optionally comprises a peptide linker between the fusion partner and the effector molecule, i.e., the linker is fused at the N- or C-terminus to the TSP-1 repeat homology domain of the CCN protein (TSP-1 domain protein / polypeptide).

[0137] Any peptide linker (as long as it is not a CCN protein sequence) may be used, many of which are known and described in the art. The linker may be a flexible linker sequence (which may include repeats of a flexible linker sequence motif). Typical linkers known in the art are rich in small non-polar (e.g., glycine) or polar (e.g., serine or threonine) residues, and generally consist of stretches of glycine and serine residues (GS), or other amino acid residues such as alanine, lysine and / or glutamic acid (A, K, and / or E), or any amino acid at all. A commonly used linker is the (GGGGS) linker (SEQ ID NO: 121), which may be provided as a repeat unit in the linker (as (GGGGS)n, where the number of copies of n is adjustable, e.g., 1-10, 1-6, 1-4, etc.). The linker may be 1-50, 1-45, 1-40, 1-30, 1-25, 1-20, 1-15, 1-12, 1-10, such as 1-8, 1-6, 1-5, or 1-4 amino acids in length. A variety of different linkers are described and used in the examples below, and any of these may be used in any of the fusion proteins of the invention.

[0138] In some embodiments, the linker comprises no more than 50 amino acids. The properties of the peptide linker may further improve the maintenance of effector function. However, the peptide linker may be susceptible to endopeptidase cleavage and removal of the fusion protein. Glycine-containing peptide linkers, with or without interspersed serine residues, are commonly utilized, but this design does not always result in fusion proteins with the desired activity and resistance to endopeptidases. In US20180273603, which discloses neurotrophin binding protein-Fc fusion proteins, the use of an α-helical linker containing repeats of the sequence A(EAAAK)A (SEQ ID NO: 14 in the document) is suggested. Furthermore, US2018 / 0127478 discloses that the use of an amino acid linker consisting of 1-3 repeats of the sequence EAAAK is suggested in Fc fusion proteins.

[0139] According to the present invention, a linker consisting of the peptide sequence EAAAK (herein SEQ ID NO: 21) may also be incorporated between the TSP-1 homology domain and the fusion partner (Fc fragment). More preferably, the linker is composed of repeats of the amino acid sequence EAAAK.

[0140] When a linker is included in the fusion protein of the present invention, the linker is disposed between the fusion partner and the effector molecule, i.e., domain III of the CCN protein. The linker may be introduced either at the C-terminus or N-terminus of domain III of the CCN protein.

[0141] Furthermore, the helical linker was resistant to endopeptidase cleavage after expression of the recombinant protein in suspension CHO cells, which is important for both manufacturing purposes and for in vivo efficacy. Furthermore, incorporation of an α-helical linker between the Fc fragment and the effector domain in an Fc fusion protein has been shown to reduce the aggregation tendency of the Fc fusion protein.

[0142] Although these findings have been demonstrated with fusion proteins comprising domain III of a CCN family protein as the effector protein, it is believed that the favorable reduced aggregation tendency and protease resistance effects may also be obtained by combining other effector molecules with the Fc fragment and α-helical linker according to the invention.

[0143] Thus, the present invention provides an Fc fusion protein comprising an Fc fragment having a peptide linker sequence of the formula aa1-aa2-(EAAAK)n-aa3-aa4-aa5, where n≧4, between the Fc fragment and the effector molecule, and aa1, aa2, aa3, aa4, aa5 are, independently, absent or one amino acid. The linker may be located at the N-terminus or C-terminus of the Fc fragment. According to one embodiment, n is 8. According to another embodiment, aa1 is threonine (T), and aa1, aa2, aa3, aa4 and aa5 are Ala (A). According to one embodiment, the linker of said Fc fusion protein is selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25. In particular, the use of fusion proteins according to the invention which include a linker consisting of (EAAAK) repeats, ie for example (EAAAK)n, where n is 8, advantageously results in less aggregation.

[0144] Another linker that may be used in accordance with the present invention is a linker (TEGRMD) having the amino acid sequence as shown in SEQ ID NO:20. In one embodiment, the invention may therefore include the incorporation of a linker peptide between the fusion partner and domain III of CCN5 (e.g., SEQ ID NO:1 to SEQ ID NO:12, 37 or 38). Non-limiting examples of fusion proteins incorporating the linker of SEQ ID NO:20 are shown in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively.

[0145] If the invention takes the aspect of domain III of CCN5 genetically fused to the N-terminus of an Fc fragment of IgG subtype IgG4 (as in SEQ ID NO: 15) incorporating a peptide linker (as in SEQ ID NO: 20) and the following mutations (S228P, F234A, L235A, K447-, Eu numbering), the complete sequence would be as in SEQ ID NO: 28, also referred to as CCN5(dIII)-Fcv2.

[0146] If the invention takes the aspect of domain III of CCN5 genetically fused to the N-terminus of an Fc fragment of IgG subtype IgG4 as shown in SEQ ID NO: 18 incorporating a peptide linker (as in SEQ ID NO: 20) and the following mutations (S228P, E233P, F234V, L235A, G236-, K447-, Eu numbering), the resulting sequence would be as shown in SEQ ID NO: 29, also referred to as CCN5(dIII)-Fcv2.1.

[0147] If the present invention takes the aspect of domain III of CCN5 genetically fused to the N-terminus of a peptide linker (as in SEQ ID NO:20) and a chimeric Fc fragment of IgG subtype IgG2 / 4 subtype as shown in SEQ ID NO:19, the resulting sequence would be as shown in SEQ ID NO:30, also referred to as CCN5(dIII)-Fcv2.3.

[0148] When the invention takes the aspect of domain III of CCN5 (as in SEQ ID NO: 1) genetically fused to the N-terminus of a peptide linker (as in SEQ ID NO: 25) and a chimeric Fc fragment of IgG subtype IgG2 / 4 subtype as shown in SEQ ID NO: 19, the resulting sequence would be as shown in SEQ ID NO: 31, also referred to as CCN5(dIII)-HLn8-Fcv2.3.

[0149] According to a preferred embodiment of the present invention: 1) a point mutation within domain III of a CCN family protein, particularly CCN5, that results in a decreased proteolytic susceptibility of domain III (see SEQ ID NO:7); 2) engineered chimeras of human IgG4 and human IgG2 Fc fragments (SEQ ID NO: 19) that have reduced proteolytic susceptibility compared to previously described Fc fragment backbones utilized in Fc fusion proteins; and 3) Optimized peptide linker compositions with reduced proteolytic susceptibility, enhanced biological activity of the fusion protein, and reduced aggregation tendency of the fusion protein (see SEQ ID NOs: 21-25) The present invention provides a fusion protein according to the present invention comprising:

[0150] In some embodiments, the peptide linker between the amino acid sequence of (i) and the monomeric fusion partner has an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-25 or 39, or an amino acid sequence having 80% sequence identity to said sequence.

[0151] Further linker sequences which may be used in accordance with the present invention are provided in SEQ ID NOs:57, 63, 65, 67 and 121. Recombinant Expression Recombinant and fusion proteins according to the invention may be produced by culturing host cells that allow the expression of the nucleotide sequence encoding said protein. Those skilled in the art are familiar with the various available biotechnological techniques that provide for the expression of isolated nucleic acid sequences for the preparation of recombinant proteins by heterologous expression in various host cell systems using commonly available genetic engineering techniques and recombinant DNA expression systems. See, for example, "Recombinant Gene Expression Protocols, Methods in Molecular Biology, 1997, edited by Rocky S Tuan, Human Press (ISSN 1064-3745), or Sambrook et al., Molecular Cloning: A laboratory Manual (3rd edition), 2001, CSHL Press, (ISBN 978-087969577-4). For example, a nucleic acid sequence encoding a recombinant protein according to the invention may be inserted into a suitable expression vector containing all the necessary transcriptional and translational control sequences specifically adapted for directing the expression of the desired protein-encoding nucleic acid sequence in a suitable host cell. Suitable expression vectors are, for example, plasmids, cosmids, viruses or artificial yeast chromosomes (YACs).

[0152] DNA sequences encoding the recombinant proteins of the present invention may be synthesized by methods well known to those skilled in the art or by commercial suppliers well known to those skilled in the art, such as Genscript, Thermo Fisher Scientific, etc.

[0153] According to one embodiment of this aspect, there is provided a DNA molecule comprising a nucleic acid sequence as set forth in SEQ ID NO: 86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112, or 113, or a sequence having at least 80% sequence identity to any of the preceding sequences. Also provided is an expression vector comprising such a DNA molecule. According to another embodiment of this aspect, there is also provided a host cell comprising such a vector.

[0154] The DNA sequence to be expressed and used to prepare a recombinant protein may be inserted into a vector commonly known as an entry vector using the Gateway cloning system (Esposito et al., 2009, "Gateway Cloning for Protein Expression", in Methods in Molecular Biology, 498, pp. 31-54). Genes cloned into an entry vector may be easily introduced into a variety of expression vectors by recombination. By way of example, synthetic sequences encoding recombinant or fusion proteins according to the invention may be recombined by BP Gateway recombinase cloning to generate an entry vector that may be used to propagate the plasmid in a suitable host cell, e.g., E. coli cells, which have been mutated to allow efficient propagation of the plasmid, e.g., One Shot Top10 TM Cells are used.

[0155] According to one embodiment of the present invention, an expression vector is prepared that contains a DNA sequence encoding a recombinant protein or fusion protein according to the present invention operably linked to a promoter. Those skilled in the art know that, as used herein, "promoter" refers to a DNA region upstream (5' end) of a DNA coding sequence that controls and initiates the transcription of a particular gene. A promoter controls the recognition and binding of RNA polymerase and other proteins to initiate transcription. "Operably linked" refers to a functional link between a promoter and a second sequence, where the promoter sequence initiates and mediates the transcription of a DNA sequence corresponding to the second sequence. Generally, "operably linked" means that the nucleic acid sequences being linked are contiguous.

[0156] Entry vectors as well as expression vectors, such as those generated from the destination vectors mentioned below, can be isolated using standard plasmid isolation techniques well known to those skilled in the art, for example from Qiagen TM QIAprep TM Spin Miniprep Kit or QIAGEN TM It may be isolated using the Plasmid Plus Maxi Kit.

[0157] When using an entry vector containing a DNA sequence encoding a recombinant or fusion protein according to the present invention, the entry vector may be further recombined with a destination vector using LR Gateway recombinase to generate an expression vector. The expression vector may then be used to express the protein-encoding DNA sequence in a suitable host cell. A non-limiting example of an applicable destination vector is pUCOE-DHFR-DEST, e.g., as described in Kaasboell et al., J. Biol. Chem, 293:46, pp. 17953-17970.

[0158] The resulting expression vector may also be verified by standard restriction enzyme digestion and DNA gel electrophoresis. According to one aspect of the present invention, there is provided an expression vector comprising a nucleic acid sequence encoding a recombinant protein of formula (I). According to yet another aspect of the present invention, there is provided an expression vector comprising a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NOs:8-12, 37, 38, 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110 and 111; and amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37 and SEQ ID NO:38, and fragments or variants thereof having at least 50% sequence identity to SEQ ID NOs:84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110 and 111.

[0159] According to another aspect, an expression vector is provided that encodes a recombinant protein comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:37, and SEQ ID NO:38, and SEQ ID NOs:84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110, and 111.

[0160] According to a further aspect of the invention there is provided an expression vector encoding a fusion protein according to the invention. Those skilled in the art are familiar with the degeneracy of the genetic code and the preference of certain codons in various organisms. Therefore, depending on the selection of the host cell, the nucleic acid sequence encoding the recombinant protein and fusion protein of the present invention may be adapted to the preferred codons of the host cell. Thus, the amino acids of the protein of the present invention may be coded by any combination of codons as shown in the table below.

[0161] [Table 1]

[0162] Preferably, the codons are further optimized for high expression according to the host cell selected. For the expression of the protein by recombinant DNA technology, in addition to certain aspects of the invention, a DNA sequence encoding a signal peptide is preferably added to the N-terminus of the protein sequence. The signal peptide may serve to direct the localization of the fusion protein during and / or after synthesis in the host cell. It may therefore be a sequence directing the secretion of the fusion protein. The use of such signal peptide sequences is well known in the art. The signal peptide may take any form, for example it may constitute the IgGk chain signal peptide or it may constitute the signal peptide from human serum albumin (SEQ ID NO: 32).

[0163] If a signal peptide from human serum albumin (SEQ ID NO:32) is added to the N-terminus of SEQ ID NO:28, the expressed protein sequence may be as shown in SEQ ID NO:33, or SEQ ID NO:85, 89, 98, 103, 107, or 111.

[0164] Furthermore, for expression of the protein by recombinant DNA techniques, in accordance with one particular embodiment of the present invention, a protein having an amino acid sequence as set forth in SEQ ID NO:33, a nucleotide sequence as set forth in SEQ ID NO:34 or SEQ ID NO:86, 90, 99, 104, 108, or 112 may be used, wherein a translation stop codon is added to the 3' end of the coding sequence.

[0165] When the invention is embodied by the nucleotide sequence in SEQ ID NO:34, as in SEQ ID NO:35 or SEQ ID NO:86, 90, 99, 104, 108, or 112, the nucleotide sequence preferably has a Kozak sequence, e.g., GCCAAC, added immediately adjacent to the 5' end of the coding sequence. The DNA sequence may further be flanked by DNA elements that allow for subcloning, such as Gateway recombinase attB sites. However, any cloning or synthetic strategy may be used to generate the DNA sequence and facilitate subcloning into an expression vector. When the DNA sequence is incorporated into a Gateway recombinase site to allow for subcloning, the nucleotide sequence may be as shown in SEQ ID NO:36 or SEQ ID NO:87, 91, 100, 105, 109, or 113.

[0166] The resulting expression vector, containing a nucleic acid sequence encoding the recombinant or fusion protein of the invention, may be introduced into a suitable host cell for production of the desired protein. A variety of commercially available or proprietary host cells may be used. For example, the expression vector may be introduced into a eukaryotic host cell, such as a CHO cell, e.g., CHO DG44 DHFR (dihydrofolate reductase - / - ) into suspension culture adapted CHO cells. Transfection of host cells with the expression vector may be carried out by methods well known to those skilled in the art, for example using electroporation.

[0167] Upon culturing the host cells in an appropriate medium, the recombinant protein or fusion protein according to the invention, encoded by the expression vector in the host cells, is produced, and the resulting protein may be collected and purified by methods well known to those of skill in the art.

[0168] The expression vector may include a signal sequence, commonly known as a "signal peptide," for secretion of the expressed protein or fusion protein into the medium. For the isolation and purification of recombinant protein secreted from cell culture medium, one or more pretreatment or clarification steps are generally used first to remove large particles and biomass.Non-limiting examples of applicable pretreatment steps are, for example, reverse osmosis, centrifugation, filtration and diafiltration, or a combination thereof.The obtained protein is then generally purified by one or more of various chromatography methods well known to those skilled in the art, such as affinity chromatography, ion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, size exclusion chromatography or other chromatography techniques, or a combination thereof.

[0169] For example, affinity chromatography may be used to detect the presence of MabSelect TM SuRe TM Media, e.g., an FPLC chromatography system, e.g., a BioRad NGC Discover fitted with a 5 mm UV flow cell TM 5ml HiTrap MabSelect on a 10 Pro system TM SuRe TM The column may be used to purify recombinant or fusion proteins expressed by suitable host cells. After loading the sample containing the protein to be purified, the column is generally washed once or more times with one or more applicable washing buffers, and then the protein is eluted using a suitable elution buffer. The obtained protein may be further purified using one or more of the chromatographic methods listed above.

[0170] It should be understood that various modifications may be introduced into the nucleic acid sequence encoding the recombinant protein of the present invention, utilizing techniques well known to those skilled in the art, such as those that enhance expression. Modifications may be introduced to adapt the coding sequence to the desired host used, to express the sequence and thus produce the recombinant protein, by using site-directed mutagenesis. Those skilled in the art are well aware of the fact that there are host-specific codons, and, as mentioned above, that adapting heterologous nucleic acid sequences to host-specific codons increases the efficiency of expression. Other modifications may also be introduced, for example, to facilitate isolation and purification, i.e., by adding sequences that code for peptides or proteins useful for such purposes. Nucleic acid sequences encoding signal peptides that provide for the secretion of the desired recombinant protein from the host cell may also be linked to the nucleic acid sequence of the present invention.

[0171] The present invention further provides host cells suitable for producing recombinant proteins or fusion proteins according to the present invention. A variety of commercially available host cells, both prokaryotic and eukaryotic, that are specifically adapted for the production of recombinant proteins may be used. Non-limiting examples of suitable host cells are, for example, CHO cells, HEK293 cells, Pichia pastoris cells, NS0 cells or E. coli cells.

[0172] Finally, the present invention also relates to the thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein and a fusion protein comprising said TSP-1 repeat homology domain for use as a drug for treating or preventing a disorder by inhibiting or counteracting the cell signaling and cell physiological functions caused by the CCN family protein.

[0173] In one aspect, the invention provides a protein as defined herein, such as a fusion protein, for use in therapy. In some aspects, the proteins, e.g., fusion proteins, may be for use in the treatment or prevention of fibrosis, or any condition exhibiting fibrosis (i.e., any fibrotic condition or disorder). Fibrosis may affect any tissue or organ, including, for example, the lung, eye, heart, skeletal muscle, peritoneum, kidney, liver, pancreas, bile duct, skin, blood vessels, or more systemic systems. In particular, the condition exhibiting fibrosis may be selected from pulmonary fibrosis of any etiology including idiopathic pulmonary fibrosis, bronchopulmonary dysplasia, retinal fibrosis, diabetic retinopathy, age-related macular degeneration, retinal detachment, oxygen-induced retinopathy, glaucoma, cardiac fibrosis, post-transplant graft fibrosis, myocardium-associated fibrosis, myofibrosis, Duchenne muscular dystrophy, peritoneal fibrosis, diabetic nephropathy, chronic kidney disease (renal fibrosis), acute kidney injury, tubulointerstitial fibrosis, chronic allograft nephropathy, hepatic fibrosis, non-alcoholic steatohepatitis, fatty liver disease, chronic pancreatitis, biliary fibrosis, keloids, scarring, systemic sclerosis, atherosclerosis, epidural fibrosis.

[0174] In the context of cardiac fibrosis, conditions to be treated or prevented may include cardiac hypertrophy and heart failure, with or without preserved ejection fraction. In a further aspect, the present invention provides a protein, e.g. a fusion protein, as defined herein, for use in treating an inflammatory or autoimmune disease. In some embodiments, the inflammatory disease is selected from rheumatoid arthritis, amyotrophic lateral sclerosis (ALS), inflammatory bowel disease, ulcerative colitis, and Crohn's disease.

[0175] In a further aspect, the present invention provides a protein, such as a fusion protein, as defined herein, for use in the treatment of cancer. In this context, it is known that 4-domain CCN proteins can induce oncogenic responses in isolated cancer cells, either directly on the cancer cells or by acting on the tumor stroma, and can also contribute to metastasis, chemoresistance and immunotherapy resistance. The ability of the proteins herein to inhibit the effect or activity of 4-domain CCN proteins thus provides a rationale for treating cancer. The cancer may be either a malignant or pre-malignant neoplastic state. The cancer may be of any tissue or organ. In one embodiment, the cancer may manifest as a solid tumor. In another embodiment, the cancer may be of or in the hematopoietic system. The cancer may be a primary or secondary cancer, or a metastasis. Thus, the cancer may be of the pancreas, breast, prostate, cervix, ovary, liver, bladder, brain, blood, bone, skin, lung or stomach. In some embodiments, the cancer is selected from pancreatic cancer, pancreatic ductal adenocarcinoma, breast cancer, prostate cancer, cervical cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, urothelial bladder cancer, brain cancer, glioblastoma, acute lymphoblastic leukemia, osteosarcoma, melanoma, mesothelioma, gastric cancer, oral squamous cell carcinoma, esophageal cancer, colorectal cancer, and lung cancer.

[0176] In a further aspect, the present invention provides a protein, such as a fusion protein, as defined herein for use in treating metabolic disease.Metabolic disease may be or may be associated with insulin resistance or glucose intolerance.In some embodiments, metabolic disease is selected from type 2 diabetes and metabolic syndrome.

[0177] The fusion proteins of the invention may also be used in methods of treatment of the above-mentioned conditions.Similarly, the fusion proteins of the invention may be used in methods of manufacture of a medicament for use in the treatment of the above-mentioned conditions. The present invention includes, but is not limited to, the following aspects. [Aspect 1] 1. A monomeric fusion protein comprising: (i) a polypeptide corresponding to at least a portion of a thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein; (ii) a monomeric fusion partner fused at the N-terminus or C-terminus to the amino acid sequence of (i); and (iii) optionally, a peptide linker between the polypeptide of (i) and the monomeric fusion partner of (ii). Including, (i) the polypeptide is 40 to 60 amino acids in length and comprises an amino acid sequence selected from SEQ ID NO: 37 or 2 to 6, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 37 or 2 to 6, and all of the cysteine ​​residues in said sequence selected from SEQ ID NO: 37 or 2 to 6 are conserved; and (ii) the monomeric fusion partner and (iii) the peptide linker are not or do not contain an IGF-binding protein homology domain, a von Willebrand factor C-type repeat homology domain, or a cysteine ​​knot domain of a CCN family protein. The monomeric fusion protein. [Aspect 2] The fusion protein of embodiment 1, wherein the polypeptide of (i) is 44 to 57 amino acids in length. [Aspect 3] (i) a polypeptide of: (a) an amino acid sequence selected from SEQ ID NO: 1 or 8-12; or (b) an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 or 8 to 12; or (c) a portion of the amino acid sequence of (a) or (b), comprising at least a 44 amino acid sequence of SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively. 3. The fusion protein of embodiment 1 or embodiment 2, comprising, or alternatively consisting of: [Aspect 4] The fusion protein according to any one of aspects 1 to 3, wherein the polypeptide consists of an amino acid sequence selected from SEQ ID NO: 37 or 2 to 6, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 37 or 2 to 6. [Aspect 5] The fusion protein of any one of embodiments 1 to 4, wherein the peptide linker of (iii) comprises no more than 50 amino acids. [Aspect 6] 6. The fusion protein of any one of aspects 1 to 5, wherein the polypeptide of (i) comprises an alanine residue at a position corresponding to position 2 of SEQ ID NO: 37 or 2 to 6, or said sequence selected from SEQ ID NOs: 1 or 8 to 12. [Aspect 7] 7. The fusion protein according to any one of aspects 1 to 6, wherein the amino acid sequence of (i) comprises an amino acid sequence selected from SEQ ID NOs: 7, 38, 42 to 46, or 47 to 51, or a sequence having at least 80% sequence identity to any of these sequences, and the protein comprises an alanine residue at a position corresponding to position 2 of said sequence in SEQ ID NOs: 7, 38, 42 to 46, or 47 to 51. [Aspect 8] The fusion protein of any one of embodiments 1 to 7, wherein said monomeric fusion partner is selected from the group consisting of serum albumin, transferrin, and a monomeric Fc fragment of human IgG. [Aspect 9] The fusion protein of embodiment 8, wherein said monomeric Fc fragment of human IgG is a monomeric Fc fragment of IgG1, IgG2, or IgG4. [Aspect 10] The fusion protein of embodiment 8 or embodiment 9, wherein the monomeric Fc fragment is aglycosylated. [Aspect 11] The fusion protein of any one of embodiments 8 to 10, wherein the monomeric Fc fragment comprises stabilizing disulfide bridges and / or protease stabilizing mutations. [Aspect 12] 12. The fusion protein of any one of embodiments 8 to 11, wherein the monomeric Fc fragment has no immune effector function. [Aspect 13] 13. The fusion protein of any one of embodiments 1 to 12, wherein the peptide linker between the amino acid sequence of (i) and the monomeric fusion partner has an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 25, 39, 57, 63, 65 or 67, or an amino acid sequence having 80% sequence identity thereto. [Aspect 14] The fusion protein of any one of embodiments 1 to 8, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110, and 111, or an amino acid sequence having 80% sequence identity thereto. [Aspect 15] A DNA molecule encoding a monomeric fusion protein as defined in any one of embodiments 1 to 14. [Aspect 16] 16. The DNA molecule of embodiment 15, further comprising a nucleotide sequence encoding a signal sequence. [Aspect 17] 17. The DNA molecule of embodiment 15 or 16, comprising a nucleotide sequence as set forth in SEQ ID NO: 34, 35, 36, 86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113, or a nucleotide sequence having at least 80% sequence identity to any of said sequences. [Aspect 18] An expression vector comprising a DNA molecule as defined in any one of embodiments 15 to 17. [Aspect 19] A host cell comprising a vector as defined in embodiment 18. [Aspect 20] 15. The fusion protein according to any one of embodiments 1 to 14 for use in therapy. [Aspect 21] The fusion protein according to any one of embodiments 1 to 14 for use in treating or preventing a disorder by inhibiting or counteracting cell signaling and cell physiological functions caused by a four domain CCN family protein. [Aspect 22] 22. The fusion protein according to any one of embodiments 1 to 14, 20 or 21 for use in the treatment or prevention of fibrosis or any condition exhibiting fibrosis. [Aspect 23] 23. The fusion protein according to any one of embodiments 1 to 14, or 20 to 22, for use in the treatment of cancer. [Aspect 24] A protein having a length of 40 to 60 amino acids, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 7, 38, 42 to 46, 47 to 51, or a sequence having at least 80% sequence identity thereto, said protein comprising an alanine residue at position corresponding to position 2 of said sequence in SEQ ID NO: 7, 38, 42 to 46, 47 to 51, and all of the cysteine ​​residues in said sequence are conserved. EXAMPLES

[0178] Example 1 Expression of fusion proteins according to the invention In this example, we describe the provision of a fusion protein (CCN5(dIII)-Fcv2) (i.e., a fusion protein according to SEQ ID NO: 28) comprising amino acids 194-246 of CCN5 (SEQ ID NO: 1) fused to the N-terminus of a peptide linker (SEQ ID NO: 20) and an Fc fragment of IgG, IgG4 subclass (S228P, F234A, L235A, K447-, Eu numbering) of SEQ ID NO: 15. An N-terminal signal sequence originating from albumin of SEQ ID NO: 32 was further added to the fusion protein, which was expressed in mammalian cells as disclosed below.

[0179] The DNA sequence shown in SEQ ID NO:36 was synthesized by a commercial supplier and the sequence was verified. The synthesized sequence was recombined with pDonrZeo by BP Gateway recombinase cloning to generate an entry vector. Competent E. coli (One Shot Top10) mutated to allow efficient propagation of the plasmid was transformed with pDonrZeo. TM After transfection of 1000 cells, Qiagen TM QIAprepTM The entry vector was isolated using standard plasmid isolation techniques through the use of a spin miniprep kit. After plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to those of skill in the art.

[0180] The destination vector was further recombined with the entry vector containing SEQ ID NO: 35 using LR Gateway recombinase. The destination vector used was pUCOE-DHFR-DEST, as described in Kaasboell et al., 2018, supra.

[0181] Competent E. coli (One Shot Top10) mutated to allow efficient propagation of plasmids TM After transfection of 1000 cells, QIAGEN TM The expression vector was isolated using standard plasmid isolation techniques using the Plasmid Plus Maxi Kit. The resulting expression vector was verified by standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to those skilled in the art. The resulting expression vector was then transformed into a plasmid using Expifectamine. TM The transfection was performed according to the "Max Titer" protocol provided by the manufacturer of the CHO transfection kit (Gibco catalog number A29129) and into CHO cells adapted to ExpiCHO suspension culture as briefly described in Kaasboell et al., 2018, supra. Six days after transfection, the cells were sedimented by centrifugation at 4750g, 4°C for 20 min, and the supernatant cell medium was harvested. 0.1 M PMSF in 100% isopropanol was added to a concentration of 1 mM, and 0.5 M EDTA was added to a concentration of 2 mM. 96% ethanol was then added to a final concentration of approximately 3%. Prior to chromatographic purification, 1 M TrisHCl pH 7.4 was added to a final concentration of 25 mM.

[0182] The capture step of the purification was performed by affinity chromatography on Protein A chromatography medium. The medium used in this experiment was rProtein A FF (GE Healthcare). From 60 mL of cell culture medium harvested and supplemented as described above, 5 mL HiTrap was used for purification of the expressed recombinant protein. TM An rProtein A FF column (GE Healthcare) was used. An FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell was used. TM 10 Pro series) on HiTrap TM An rProtein A FF column was mounted and equilibrated with a buffer containing 25 mM TrisHCl pH 7.4, 25 mM NaCl and 3% ethanol. The harvested cell medium containing the recombinant protein was loaded using a sample pump at a rate of 2.5 ml / min, then washed with 6 column volumes of wash buffer (25 mM TrisHCl pH 7.4, 25 mM NaCl and 3% ethanol) and then eluted with 0.1 M Na citrate, pH 3.0 in 3% ethanol. Elution with UV 280 nm absorbance above 100 mAU was collected in 3 mL fractions in low protein binding tubes pre-filled with 1 mL 1 M TrisHCl pH 9.0. Fractions containing the UV absorbance peak were concentrated to 500 μL using a Vivaspin® 20 mL, 30 kDa MWCO concentration device. After concentration, the elution was performed using an FPLC chromatography system (BioRad NGC Discover TMThe sample was loaded into the sample loading loop on a FPLC chromatography system (GE Healthcare 10 Pro system). The FPLC chromatography system was equipped with a Superdex® 200 Increase 10 / 300 GL column (GE Healthcare) equilibrated with 50 mM NaCl, 20 mM HEPES pH 7.0. The sample was injected and the column was perfused with pre-equilibration buffer (50 mM NaCl, 20 mM HEPES pH 7.0) at a flow rate of 0.25 mL / min. The major UV 280 nm absorbance peak was found to contain the purified recombinant protein (CCN5(dIII)-Fcv2, SEQ ID NO:28). A 10 μL sample of the collected fractions was transferred to a Mini-PROTEAN® TGX Stain-Free TM The gels were subjected to SDS-PAGE using precast gels and analyzed by ChemiDoc. TM The isolated recombinant proteins were visualized using an imaging system (BioRad).

[0183] It is widely known to those skilled in the art that recombinant proteins can be produced in a variety of expression systems and purified by a variety of chromatographic methods with similar results.

[0184] Example 2 To produce a recombinant protein according to SEQ ID NO:28, a DNA sequence was expressed encoding a fusion protein (CCN5(dIII)-Fcv2) comprising amino acids 194-246 of CCN5 (SEQ ID NO:1) fused to the N-terminus of a peptide linker (SEQ ID NO:20) and an Fc fragment of IgG, subclass IgG4 (S228P, F234A, L235A, K447-, Eu numbering) of SEQ ID NO:15.

[0185] The resulting proteins were tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells (Figure 1A). Tissue culture-treated Corning Incorporated Costar® 96-well sterile polystyrene plates were coated with fibronectin (Sigma catalog number F1141) diluted to 10 μg / mL in Dulbecco's Phosphate Buffered Saline (Lonza catalog number 17-512F, hereafter referred to as PBS) from BioWhittaker®. A coating solution containing fibronectin was dispensed into the wells in a volume of 100 μL / well and after 1 hour of incubation at room temperature, the coating solution was decanted and 100 μL of PBS was also dispensed into the fibronectin-coated wells and this was also decanted. A549 cells, subcultured to maintain a density of up to 80% confluence, were detached by enzymatic treatment (Accutase®, Biowest® Catalog No. L0950-100), diluted to a concentration of 110000 cells / mL in Dulbecco's Modified Eagle Medium with High Glucose (Gibco Catalog No. 41965-039) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (FBS equilibrated to room temperature (Gibco Catalog No. 16000-044) was incubated in a water bath for 30 minutes with shaking) and 50 μg / ml gensumycin (Sanofi), and 100 μL of the cell solution was distributed into fibronectin-coated wells. All cell incubations were performed in a cell culture incubator maintained at a temperature of 37°C, room air humidified atmosphere and 5% CO2. After overnight incubation, A594 cells were washed twice with PBS and 90 μL of Dulbecco's Modified Eagle Medium without FBS and high glucose (DMEM, Gibco Cat. No. 41965-039) and 50 μg / ml gensmycin (Sanofi) were dispensed into the wells. After 18 hours of incubation in FBS-free medium, cells were stimulated with 10 μL of a solution of the recombinant protein of interest.After 60 min of stimulation, the cells were harvested by decanting the medium and adding 50 μL lysis buffer containing blocking reagent as supplied by the Cisbio phospho-AKT (Ser473) kit (Cisbio Inc, Cat. No. 64KSPEG). After adding lysis buffer containing blocking reagent, the 96-well plate is incubated for 60 min on a PST-60HL plus (ThermoFisher) plate shaker at 500 rpm. After shaking, the lysed samples were tritrurated and then transferred 16 μL from each well to a white-walled HTRF 96-well low volume plate (Cisbio Inc., Cat. No. 66PL96025). To assay the amount of phosphorylated AKT (Ser473), 4 μL of labeled antibody mixture (50 / 50 volume / volume mixture of phospho-AKT d2 and phospho-AKT cryptate from Cisbio Inc, Cat. No. 64AKSPEG) was added to each well (negative control wells used only cryptate antibody), the plate was sealed with adhesive plastic film and after overnight incubation at 4° C. read on a PolarStar Omega plate reader (BMG Labtech, Germany) equipped with a TR-FRET recording head and 337 nm emission and 615 nm and 665 nm excitation filters. The ratio between the 665 nm and 615 nm excitation recordings was blank corrected and the values ​​of wells stimulated with recombinant protein were expressed as a percentage of wells stimulated with vehicle.

[0186] Example 3 To produce a recombinant protein according to SEQ ID NO:28, a DNA sequence was expressed encoding a fusion protein (CCN5(dIII)-Fcv2) comprising amino acids 194-246 of CCN5 (SEQ ID NO:1) fused to the N-terminus of a peptide linker (SEQ ID NO:20) and an Fc fragment of IgG subclass IgG4 of SEQ ID NO:15 (S228P, F234A, L235A, K447-, Eu numbering).

[0187] The resulting proteins were tested for their ability to inhibit TGF-β-stimulated profibrotic transcription (transcription from SMAD2 / 3-bound cis-elements) in IMR90 human lung fibroblasts (Figure 1D). The assay was performed technically as described above in Kaasboell et al. (2018) except that 2500 IMR90 lung fibroblasts / well were utilized instead of Rat2 cells. Proteins used for stimulation were as shown in Figure 1D. Prior to use, IMR90 cells were subcultured as described above for A549 cells. IMR90 cells were used prior to passage 20, i.e., before reaching replicative senescence.

[0188] Example 4 To produce a recombinant protein according to SEQ ID NO:28, a DNA sequence was expressed encoding a fusion protein (CCN5(dIII)-Fcv2) comprising amino acids 194-246 of CCN5 (SEQ ID NO:1) fused to the N-terminus of a peptide linker (SEQ ID NO:20) and an Fc fragment of IgG, subclass IgG4 (S228P, F234A, L235A, K447-, Eu numbering) of SEQ ID NO:15.

[0189] The resulting proteins were tested for their ability to inhibit the proliferation of the human lung fibroblast cell line IMR90 (Figure 1B). Before use, IMR90 cells were subcultured as described above for A549 cells. IMR90 cells were used before passage 20, i.e. before reaching replicative senescence. For the experiments, IMR90 cells were harvested as described above for A549 cells, washed in PBS, diluted in DMEM with 1% FBS containing gensmicin as described above for Example 2, and seeded in xCELLigence impedance plates at a density of 12000 / well. After 2 hours, the cells were stimulated with a 10 μL solution of the recombinant protein in question or FBS, and after a further 72 hours of incubation, harvested with CellTiter-Glo® (Promega Inc.) as described above in Kaasboell et al. (2018), supra.

[0190] Example 5 To produce a recombinant protein according to SEQ ID NO:28, a DNA sequence was expressed encoding a fusion protein (CCN5(dIII)-Fcv2) comprising amino acids 194-246 of CCN5 (SEQ ID NO:1) fused to the N-terminus of a peptide linker (SEQ ID NO:20) and an Fc fragment of IgG, subclass IgG4 (S228P, F234A, L235A, K447-, Eu numbering) of SEQ ID NO:15.

[0191] The resulting proteins were tested for their ability to inhibit the sphere-forming (anchorage-independent growth) ability of the estrogen receptor-positive breast cancer cell line MCF-7 and the triple-negative breast cancer cell line MDA-MB-231 as described in Kaasboell et al., supra (Figure 1C). MDA-MB-231 cells were treated the same as described for the MCF-7 cell line in Kaasboell et al., supra. MCF-7 and MDA-MB-231 cell lines were subcultured as described for the A549 cell line above.

[0192] Example 6 To produce recombinant proteins according to SEQ ID NO: 28 (CCN5(dIII)-Fcv2), SEQ ID NO: 29 (CCN4(dIII)-Fcv2.1) and SEQ ID NO: 30 (CCN5(dIII)-Fcv2.3), a peptide linker (SEQ ID NO: 20) and an IgG of SEQ ID NO: 15, an Fc fragment of subclass IgG4 (S228P, F234A, L235A, K447-, Eu numbering) (CCN5(dIII)-Fcv2), sequence DNA sequences encoding fusion proteins containing amino acids 194 to 246 of CCN5 (SEQ ID NO: 1) fused to the N-terminus of an IgG numbered 18, Fc fragment of subclass IgG4 (S228P, F233P, F234V, L235A, G236-, K447-, Eu numbering) (CCN5(dIII)-Fcv2.1) or a chimeric Fc fragment of IgG2 / 4 subclass (SEQ ID NO: 19) (CCN5(dIII)-Fcv2.3) were expressed.

[0193] In particular, expression vectors encoding for the expression of SEQ ID NO:28 (CCN5(dIII)-Fcv2), SEQ ID NO:29 (CCN4(dIII)-Fcv2.1) and SEQ ID NO:30 (CCN5(dIII)-Fcv2.3) were synthesized using Expifectamine TM Transfections were performed into ExpiCHO suspension culture adapted CHO cells according to the "Max Titer" protocol provided by the manufacturer of the CHO transfection kit (Gibco catalogue no. A29129) and as briefly described in Kaasboell et al., supra. Six days after transfection, cells were pelleted by centrifugation at 13000 rpm for 5 minutes in a Heraeus biofuge pico benchtop centrifuge and the supernatant cell medium was harvested. Samples of the harvested cell culture supernatant were stained with Mini-PROTEAN® TGX Stain-Free TM Separation was performed by SDS-PAGE using precast gels and ChemiDoc TM The recombinant proteins were visualized using an imaging system (BioRad). The separated proteins were then transferred to a PVDF membrane for Western blot analysis using the Trans-Blot Turbo, semi-dry blotting system (Bio-Rad). For visualization, SuperSignal TM West Femto Maximum sensitive substrate (ThermoFisherScientific) and ChemiDoc TM Blots were probed with an anti-human IgG4 antibody conjugated to horseradish peroxidase (Invitrogen Catalog No. A10654) used in conjunction with an imaging system (BioRad).

[0194] In FIG. 2, data are presented demonstrating the improved protease resistance of an Fc fragment backbone constructed with an IgG2 / 4 chimera (set forth in SEQ ID NO: 19). Either CCN5 / WISP2 (domain III) fused to an IgG4 Fc fragment containing an immune effector silenced IgG4 hinge (as defined in SEQ ID NO: 28); CCN5 (domain III)-Fcv2, the same IgG4 backbone incorporating IgG2-based mutations (as defined in SEQ ID NO: 29); CCN5 (domain III)-Fcv2.1, or the same IgG4 backbone with a complete hinge region from IgG2 (as defined in SEQ ID NO: 30); CCN5 (domain III)-Fcv2.3 were expressed in the ExpiCHO system and conditioned medium (CM) was harvested after 6 days. Western blotting and total protein staining of SDS-PAGE gels reveal that the CCN5 (domain III)-Fcv2.3 variant is the least sensitive to proteases present in the culture. Note that the immunoreactivity of anti-IgG4 antibodies against the Fc fragment is partially lost upon substitution of IgG2-derived sequences and therefore underestimates protein levels compared to general protein staining.

[0195] Example 7 To produce recombinant proteins according to SEQ ID NO:30 (CCN5(dIII)-Fcv2.3) and SEQ ID NO:31 (CCN5(dIII)-HLn8-Fcv2.3), DNA sequences were expressed that encoded fusion proteins comprising amino acids 194-246 of CCN5 (SEQ ID NO:1) fused to the N-terminus of either the peptide linker and chimeric Fc fragment of IgG2 / 4 subclass (SEQ ID NO:19) set forth in SEQ ID NO:20 (CCN5(dIII)-Fcv2.3) or the peptide linker and chimeric Fc fragment of IgG2 / 4 subclass (SEQ ID NO:19) set forth in SEQ ID NO:25 (CCN5(dIII)-HLn8-Fcv2.3).

[0196] In particular, expression vectors encoding for the expression of SEQ ID NO: 30 (CCN5(dIII)-Fcv2.3) and SEQ ID NO: 31 (CCN5(dIII)-HLn8-Fcv2.3) were synthesized using Expifectamine TMTransfections were performed into ExpiCHO suspension culture adapted CHO cells according to the "Max Titer" protocol provided by the manufacturer of the CHO transfection kit (Gibco Cat. No. A29129) and as briefly described in Kaasboell et al., supra. Four days after transfection, cells were pelleted by centrifugation at 13000 rpm for 5 min in a Heraeus biofuge pico benchtop centrifuge and the supernatant cell medium was harvested. Samples of the harvested cell culture supernatant were stained with Mini-PROTEAN® TGX Stain-Free TM Precast gels were used for separation by SDS-PAGE. Separated proteins were transferred to PVDF membranes for Western blot analysis using the Trans-Blot Turbo semi-dry blotting system (Bio-Rad). For visualization, SuperSignal was used. TM West Femto Maximum sensitive substrate (ThermoFisherScientific) and ChemiDoc TM Blots were probed with an anti-human IgG4 antibody conjugated to horseradish peroxidase (Invitrogen catalog no. A10654) used in conjunction with an imaging system (BioRad).

[0197] In FIG. 3, data are provided showing the trend towards reduced aggregation when an embodiment of the invention incorporates the peptide linker shown in SEQ ID NO:25. Non-reducing SDS-PAGE of CM from transiently transfected CHO suspension cells expressing CCN5 (domain III) fused to the amino terminus of chimeric IgG2 / 4 Fc fragment through various peptide linkers. Western blot reveals that the fusion protein (dIII)-HLn8-Fcv2.3 comprising the amino acid sequence as shown in SEQ ID NO: 31 has a lower tendency to aggregate than the fusion protein of the present invention CCN (domain III)-Fcv2.3 having the amino acid sequence as shown in SEQ ID NO: 30. This finding indicates that the peptide linker defined in the sequence of SEQ ID NO: 25 provides a lower tendency of fusion protein aggregation compared to the fusion protein containing the peptide linker defined in the sequence of SEQ ID NO: 20.

[0198] Example 8 To produce recombinant proteins according to SEQ ID NO: 40 (Fc-HLn8-CCN5(dIII)) or SEQ ID NO: 41 (Fc-HLn8-CCN5(dIII)-P195A), DNA sequences were expressed encoding fusion proteins comprising either amino acids 194-246 of CCN5 (SEQ ID NO: 1) or amino acids 194-246 of CCN5 with amino acid 195 (proline) substituted with alanine (SEQ ID NO: 7) fused to the C-terminus of a peptide linker (SEQ ID NO: 39) and an Fc fragment of IgG subtype IgG4 of SEQ ID NO: 15 (S228P, F234A, L235A, K447-, Eu numbering).

[0199] In particular, expression vectors encoding for the expression of SEQ ID NO: 40 (Fc-HLn8-CCN5(dIII)) and SEQ ID NO: 41 (Fc-HLn8-CCN5(dIII)-P195A) were synthesized using Expifectamine TMTransfections were performed into ExpiCHO suspension culture adapted CHO cells according to the "Max Titer" protocol provided by the manufacturer of the CHO transfection kit (Gibco Cat. No. A29129) and as briefly described in Kaasboell et al., supra. Three days after transfection, cells were pelleted by centrifugation at 13000 rpm for 5 minutes in a Heraeus biofuge pico benchtop centrifuge and the supernatant cell medium was harvested. Samples of the harvested cell culture supernatant were stained with Mini-PROTEAN® TGX Stain-Free TM Precast gels were used for separation by SDS-PAGE. Separated proteins were transferred to PVDF membranes for Western blot analysis using the Trans-Blot Turbo semi-dry blotting system (Bio-Rad). For visualization, SuperSignal was used. TM West Femto Maximum sensitive substrate (ThermoFisherScientific) and ChemiDoc TM Blots were probed with an anti-human IgG4 antibody conjugated to horseradish peroxidase (Invitrogen catalog no. A10654) used in conjunction with an imaging system (BioRad).

[0200] In FIG. 5, embodiments of the invention present data showing reduced susceptibility to endopeptidase cleavage when they incorporate a mutation of proline 195 of the CCN5 TSP-1 repeat homology domain, as shown in SEQ ID NO:7.

[0201] Reducing SDS-PAGE of CM from transiently transfected CHO suspension cells expressing CCN5 (domain III) fused to the carboxyl terminus of an IgG4 Fc fragment as set forth in SEQ ID NO: 15, either incorporating the P195A mutation (Fc-HLn8-CCN5(dIII)-P195A) or expressing the wild-type P195 variant of the CCN5 TSP-1 repeat homology domain (Fc-HLn8-CCN5(dIII)). The blot demonstrates that the P195A mutation confers proteolytic resistance to the TSP-1 repeat homology domain of CCN5.

[0202] Example 9 Disclosed is a fusion protein comprising amino acids 194-250 of human CCN5 (SEQ ID NO:56) fused to the N-terminus of a peptide linker (SEQ ID NO:57) and a human IgG, IgG4 subclass Fc fragment (S228P, F234A, L235A, K447-, Eu numbering) of SEQ ID NO:15, resulting in a protein sequence corresponding to SEQ ID NO:58 (CCN5(dIII)-SL-Fcv0). The fusion protein was further supplemented with an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO:32, resulting in a fusion protein corresponding to SEQ ID NO:59, and expressed in mammalian cells as disclosed below.

[0203] The DNA sequence encoding the fusion protein of SEQ ID NO:59 was codon-optimized (according to the algorithm of the commercial supplier) for protein expression in hamster cells, and a KOZAK sequence for translation was added at the 5' end and a stop codon was introduced at the 3' end to generate the DNA sequence of SEQ ID NO:60. Gateway attB sites were further added at both ends of the DNA sequence to generate the DNA sequence of SEQ ID NO:61. The sequence of SEQ ID NO:61 was synthesized and verified by the commercial supplier. The synthesized sequence was recombined with pDonrZeo by BP Gateway recombinase cloning to generate the entry vector. Competent E. coli (One Shot Top10) mutated to allow efficient propagation of the plasmid was transformed into TMAfter transfection of 1000 cells, Qiagen TM QIAprep TM The entry vector was isolated using standard plasmid isolation techniques through the use of a spin miniprep kit. After plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to those of skill in the art.

[0204] The entry vector containing the sequence of SEQ ID NO: 60 was further recombined with the destination vector using LR Gateway recombinase. The destination vector used was pUCOE-DHFR-DEST, as described in Kaasboell et al., 2018, J. Biol. Chem, 293:46, pp. 17953-17970.

[0205] Competent E. coli (One Shot Top10) mutated to allow efficient propagation of plasmids TM After transfection of 1000 cells, QIAGEN TM The expression vector was isolated using standard plasmid isolation techniques using the Plasmid Plus Maxi Kit. The resulting expression vector was verified by standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to those skilled in the art. The resulting expression vector was then transformed into a plasmid using Expifectamine. TM Transfection was performed into ExpiCHO cells adapted to suspension culture according to the "Max Titer" protocol provided by the manufacturer of the CHO transfection kit (Gibco catalog number A29129) and as briefly described in Kaasboell et al., 2018, supra. Four days after transfection, cells were sedimented by centrifugation at 4750xg, 4°C for 20 min, and the supernatant cell medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 1 mM, and 0.5M EDTA was added to a concentration of 2 mM. 96% ethanol was then added to a final concentration of approximately 3%. Prior to chromatographic purification, 1M TrisHCl pH 7.4 was added to a final concentration of 25 mM.

[0206] Proteins were purified by affinity chromatography using Protein A chromatography media. The chromatography media used in this experiment was rProtein A FF (GE Healthcare). From 120 mL of cell culture medium harvested and replenished as described above, 5 mL HiTrap was used for purification of expressed recombinant proteins. TM An rProtein A FF column (GE Healthcare) was used. An FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell was used. TM 10 Pro series) on HiTrap TM The rProtein A FF column was loaded and equilibrated with a buffer containing 25 mM TrisHCl pH 7.4, 25 mM NaCl and 3% ethanol. The harvested cell media containing the recombinant protein was loaded using a sample pump at a rate of 2.5 ml / min, then washed with 10 column volumes of wash buffer (25 mM TrisHCl pH 7.4, 25 mM NaCl and 3% ethanol) and then eluted with 0.1 M Na citrate, pH 3.0 in 3% ethanol. 3 mL elution fractions were collected in low protein binding tubes pre-filled with 1 mL 1 M TrisHCl pH 9.0. Protein elution was monitored by 280 nm UV absorbance, and 10 μL samples of pooled fractions containing the UV 280 nm absorbance peak were purified using Mini-PROTEAN® TGX Stain-Free with or without the reducing agent β-mercaptoethanol. TM The gels were subjected to SDS-PAGE using precast gels and analyzed by ChemiDoc. TM The isolated recombinant proteins were visualized using an imaging system (BioRad).

[0207] It is widely known to those skilled in the art that recombinant proteins may be produced in a variety of expression systems and purified by a variety of chromatographic methods with similar results.

[0208] In Figure 6, expression and purification of the protein corresponding to SEQ ID NO:58 in the absence of the reducing agent β-mercaptoethanol results in a protein that migrates higher than expected, thus indicating dimer formation. However, in the presence of the reducing agent β-mercaptoethanol, expression and purification of the protein corresponding to SEQ ID NO:58 results primarily in truncated fragments and not intact protein, as can be seen from the lane containing the purified protein.

[0209] Example 10 In an attempt to increase the proteolytic resistance of the protein corresponding to SEQ ID NO: 58, a number of mutants of the sequence of SEQ ID NO: 58 were generated. DNA sequences were synthesized and verified by a commercial supplier and then subcloned to generate plasmids as described in Example 9 and proteins expressed as described in Example 9. The mutants included proteins containing modifications as listed below: 1) The N-terminal signal sequence resulting from albumin of SEQ ID NO: 32 is ligated to the amino terminus of a fragment of CCN5 consisting of amino acids 194 to 249 incorporating a mutation (P245L) corresponding to SEQ ID NO: 62, combined with a partial truncation of the peptide linker corresponding to SEQ ID NO: 63 and an Fc fragment of SEQ ID NO: 15, resulting in a sequence corresponding to SEQ ID NO: 64; 2) The N-terminal signal sequence resulting from albumin of SEQ ID NO: 32 is ligated to the amino terminus of a fragment of CCN5 consisting of amino acids 194 to 246 corresponding to SEQ ID NO: 1, combined with a peptide linker variant corresponding to SEQ ID NO: 65 and an Fc fragment of SEQ ID NO: 15, resulting in a sequence corresponding to SEQ ID NO: 66; 3) The N-terminal signal sequence resulting from albumin of SEQ ID NO: 32 is ligated to the amino terminus of a fragment of CCN5 consisting of amino acids 194 to 246 corresponding to SEQ ID NO: 1, combined with a peptide linker variant corresponding to SEQ ID NO: 67 and an Fc fragment of SEQ ID NO: 15, resulting in a sequence corresponding to SEQ ID NO: 68; 4) The N-terminal signal sequence resulting from albumin of SEQ ID NO:32 is ligated to the amino terminus of a fragment of CCN5 consisting of amino acids 194 to 246 corresponding to SEQ ID NO:1, combined with a modified peptide linker corresponding to SEQ ID NO:65 and an Fc fragment of SEQ ID NO:19, resulting in a sequence corresponding to SEQ ID NO:69.

[0210] These iterations of the protein disclosed in Example 12 (1-4 above) showed some improvement in resistance to proteolytic cleavage during expression in the ExpiCHO system performed as described in Example 9. However, expression of the proteins corresponding to SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:69 revealed that the degree of proteolytic resistance was still insufficient to allow production of intact purified protein.

[0211] Example 11 A fusion protein was generated comprising amino acids 194-237 (SEQ ID NO:38) of CCN5, with the amino acid at position 195 (proline) substituted with alanine, fused to the N-terminus of a chimeric Fc fragment (SEQ ID NO:19) of IgG subtype IgG2 / 4, including a peptide linker (SEQ ID NO:21) and a deletion of the carboxyl terminal K477-(EU numbering), resulting in SEQ ID NO:27. The fusion protein was further supplemented with an N-terminal signal sequence for secretion resulting from albumin, SEQ ID NO:32, resulting in a fusion protein corresponding to SEQ ID NO:70. The DNA sequence encoding the fusion protein, SEQ ID NO:70, was codon-optimized (according to the commercial supplier's algorithm) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced to the 3' end, resulting in the DNA sequence, SEQ ID NO:71. Gateway attB sites were further added to both ends of the DNA sequence, resulting in the DNA sequence, SEQ ID NO:72.

[0212] The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate a plasmid as described in Example 9, and the protein corresponding to SEQ ID NO:70 was expressed by transient transfection of ExpiCHO cells as described in Example 9.

[0213] Six days after transfection, cells were pelleted by centrifugation at 4750xg for 20 minutes at 4°C, and the supernatant cell medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. Prior to chromatographic purification, 1M Na citrate pH 5.5 was added to a final concentration of 30 mM.

[0214] 1mL HiTrap TM MabSelectSuRe TM Capture step on column (GE Healthcare) followed immediately by BioScale TM Proteins were purified by tandem chromatography consisting of desalting on a Mini Bio-Gel® P-6 10 mL column (BioRad). An FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell was used. TM A column was mounted on the first column switching valve. TM The column was loaded and equilibrated with a buffer consisting of 30 mM Na citrate pH 5.5, while the Bio-Gel column was loaded onto the second column switching valve and equilibrated with Buffer A2 (100 mM NaH2PO4 / Na2HPO4 pH 6.5). With the second column switching valve containing the Bio-Gel column set to bypass, 140 mL of harvested cell media containing the recombinant protein was pumped into the MabSelectSuRe column using the sample pump at a rate of 2.0 ml / min. TMAfter loading onto the column, it was washed with 5 column volumes of wash buffer A1 (30 mM Na citrate pH 5.5), followed by 5 column volumes of wash buffer A3 (30 mM Na citrate, 0.5 M NaCl, pH 5.5), followed by 3 column volumes of wash buffer A1. A Bio-Gel® column mounted on a second column switching valve was switched into the flow path before elution with elution buffer (30 mM citric acid, pH 3.4). After elution with 2 mL elution buffer, MabSelectSuRe TM The column was switched out of the flow path and purified protein was eluted from the Bio-Gel® column with Buffer A2. Protein elution was monitored by 280 nm UV absorbance and collection was triggered when absorbance exceeded 100 mAU. Collected fractions were pooled and 10 μL samples were purified with Mini-PROTEAN® TGX Stain-Free in the presence of the reducing agent β-mercaptoethanol. TM Precast gels were subjected to SDS-PAGE and analyzed by ChemiDoc TM The isolated recombinant proteins were visualized using an imaging system (BioRad).

[0215] FIG. 7 shows that expression and purification of the protein corresponding to SEQ ID NO:27, in which the carboxyl-terminal tail of CCN5 is truncated, is substantially more proteolytically resistant than mutants in which all of the carboxyl-terminal amino acids of CCN5 are included (such as in SEQ ID NOs:58, 64, 66, 68 and 69), even when the cell culture medium was harvested after an additional 2 days of subculture compared to Example 9.

[0216] Example 12 A fusion protein was generated comprising amino acids 206-249 (SEQ ID NO:44) of CCN3, with the amino acid at position 207 (isoleucine) replaced by alanine, fused to the N-terminus of a chimeric Fc fragment (SEQ ID NO:19) of IgG subtype IgG2 / 4, including a peptide linker (SEQ ID NO:21) and a deletion of the carboxyl terminal K477-(Eu numbering), resulting in a fusion protein of SEQ ID NO:73. The fusion protein was further supplemented with an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO:32, resulting in a fusion protein corresponding to SEQ ID NO:74. The DNA sequence encoding the fusion protein of SEQ ID NO:74 was codon-optimized (according to the commercial supplier's algorithm) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID NO:75. Gateway attB sites were further added to both ends of the DNA sequence, resulting in a DNA sequence of SEQ ID NO:76.

[0217] The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate a plasmid as described in Example 9, and the protein corresponding to SEQ ID NO:74 was expressed by transient transfection of ExpiCHO cells as described in Example 9.

[0218] Five days after transfection, cells were pelleted by centrifugation at 4750xg for 20 minutes at 4°C and the supernatant cell medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. Prior to chromatographic purification, 1M Na citrate pH 5.5 was added to a final concentration of 30 mM.

[0219] 5mL HiTrap TM MabSelectSuRe TM Capture step on column (GE Healthcare) followed immediately by 53mL HiPrep TMProteins were purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare) and a FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell. TM A column was mounted on the first column switching valve. TM The column was loaded and equilibrated with a buffer consisting of 30 mM Na citrate pH 5.5 while the HiPrep TM The column was mounted on a second column switching valve and equilibrated with buffer A2 (100 mM NaH2PO4 / Na2HPO4 pH 6.5). TM With the second column switching valve containing the column set to bypass, 260 mL of harvested cell media containing the recombinant protein was pumped through the MabSelectSuRe column at a rate of 3.5 mL / min using the sample pump. TM After loading onto the column, it was washed with 5 column volumes of wash buffer A1 (30 mM Na citrate pH 5.5), then 5 column volumes of wash buffer A3 (30 mM Na citrate, 0.5 M NaCl, pH 5.5), then 2 column volumes of wash buffer A1. The HiPrep loaded onto the second column switching valve was then washed with elution buffer (30 mM citric acid, pH 3.4) before elution with elution buffer (30 mM citric acid, pH 3.4). TM The column was set and entered into the flow path. After elution with 10 mL of elution buffer, MabSelectSuRe TM The column was switched out of the flow path and the purified protein was purified with Buffer A2 in HiPrep TM Protein elution was monitored by UV absorbance at 280 nm, and collection was triggered when absorbance exceeded 100 mAU. Collected fractions were pooled, and 10 μL samples were stained with Mini-PROTEAN® TGX Stain-Free in the presence or absence of the reducing agent β-mercaptoethanol. TM Precast gels were subjected to SDS-PAGE and analyzed by ChemiDoc TM The isolated recombinant proteins were visualized using an imaging system (BioRad).

[0220] In FIG. 8, it is shown that fusion proteins containing amino acids from CCN3 / Nov (domain III / TSP-1 homology domain) as disclosed in SEQ ID NO:73, as well as fusion proteins containing amino acids from homologous CCN5 (domain III / TSP-1 homology domain) as disclosed in SEQ ID NO:27, have similar or better proteolytic resistance than fusion proteins containing amino acids from CCN5, as described in Example 11 and shown in FIG.

[0221] Example 13 A fusion protein comprising amino acids 194-246 (SEQ ID NO: 7) of CCN5, with the amino acid at position 195 (proline) substituted with alanine, fused to the C-terminus of a peptide linker (SEQ ID NO: 39) and an Fc fragment of IgG, IgG4 subclass (S228P, F234A, L235A, K447-, Eu numbering) of SEQ ID NO: 15, resulting in a protein sequence corresponding to SEQ ID NO: 41, was added with an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO: 32, resulting in a fusion protein corresponding to SEQ ID NO: 77. The DNA sequence encoding the fusion protein of SEQ ID NO: 77 was codon-optimized (according to the algorithm of the commercial supplier) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID NO: 78. Gateway attB sites were further added to both ends of the DNA sequence, resulting in a DNA sequence of SEQ ID NO: 79.

[0222] The sequence of SEQ ID NO: 79 was synthesized by a commercial supplier and the sequence was verified. The synthesized sequence was recombined with pDonrZeo by BP Gateway recombinase cloning to generate an entry vector. Competent E. coli (One Shot Top10) mutated to allow efficient propagation of the plasmid was transformed with pDonrZeo. TM After transfection of 1000 cells, Qiagen TM QIAprep TMThe entry vector was isolated using standard plasmid isolation techniques through the use of a spin miniprep kit. After plasmid isolation, it was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to those of skill in the art.

[0223] The entry vector containing the sequence of SEQ ID NO: 78 was further recombined with the destination vector using LR Gateway recombinase. The destination vector used was pUCOE-DHFR-DEST, as described in Kaasboell et al., 2018, J. Biol. Chem, 293:46, pp. 17953-17970.

[0224] Competent E. coli (One Shot Top10) mutated to allow efficient propagation of plasmids TM After transfection of 1000 cells, QIAGEN TM The expression vector was isolated by standard plasmid isolation techniques using the Plasmid Plus Maxi Kit. The resulting expression vector was verified by standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to those skilled in the art. The resulting expression vector was then transfected into suspension-adapted DG44 CHO cells by electroporation using the Neon transfection system (ThermoFisherScientific). The cells were maintained in vented Erlenmeyer flasks in a cell culture incubator maintained at 37°C with 8% CO2 on a shaking platform (as described in Kaasboell et al., supra). The transfected cells were maintained overnight in CD DG44 cell medium (Gibco, Cat. No. 12610-010) and then transfected with HyClone 1000 ng / ml. TM ActiPro TMThe cells were transferred to medium (hypoxanthine and thymidine free, GE Healthcare) and subcultured until viability approached 80%, at which point the medium was supplemented with 0.1 μM methotrexate. After the addition of 0.1 μM methotrexate, the cells were again subcultured until viability approached 80%, at which point the medium was supplemented with 1 μM methotrexate. The cells were again subcultured until viability exceeded 98% and the doubling time decreased to less than 26 hours, at which point the cell pool was considered stably transfected. Once a stable cell pool was established, the cell culture volume was expanded and cultured for 1 h. * A density of 10^6 cells / mL allowed seeding of stably transfected cells for production. After 3 days of subculture, daily cultures were incubated with 4 / 0.4% v / v HyClone TM Cell Boost TM 7a / 7b were supplemented to the cell culture. After 10 days, the cells were sedimented by centrifugation at 4750xg, 4°C for 20 min, and the supernatant cell medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1M Na citrate pH 5.5 was added to a final concentration of 30 mM prior to chromatographic purification.

[0225] 5mL HiTrap as described in Example 15 TM MabSelectSuRe TM Capture step on column (GE Healthcare) followed immediately by 53mL HiPrep TM Protein was purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare).Purified protein preparation (not showing any signs of proteolytic processing) was then tested for its ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2.As can be seen in Figure 9, the purified protein corresponding to SEQ ID NO: 41 produced from a stable transfection pool of CHO suspension cells surprisingly did not show any signs of ability to inhibit phosphorylation of AKT (serine 473).

[0226] Example 14 A fusion protein was generated that contains amino acids 206-249 (SEQ ID NO: 44) of CCN3, with the amino acid at position 207 (isoleucine) replaced by alanine, fused to the N-terminus of a peptide linker (SEQ ID NO: 22) and a chimeric Fc fragment of IgG subtype IgG2 / 4 (SEQ ID NO: 19), resulting in a protein sequence corresponding to SEQ ID NO: 80, and an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO: 32 was added to yield a fusion protein corresponding to SEQ ID NO: 81. The DNA sequence encoding the fusion protein of SEQ ID NO: 81 was codon-optimized (according to the commercial supplier's algorithm) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end to yield the DNA sequence of SEQ ID NO: 82. Gateway attB sites were further added to both ends of the DNA sequence to yield the DNA sequence of SEQ ID NO: 83.

[0227] The sequence of SEQ ID NO:83 was synthesized by a commercial supplier and the sequence was verified. The synthesized sequence was recombined with pDonrZeo by BP Gateway recombinase cloning to generate an entry vector. Competent E. coli (One Shot Top10) mutated to allow efficient propagation of the plasmid was transformed with pDonrZeo. TM After transfection of 1000 cells, Qiagen TM QIAprep TM The entry vector was isolated using standard plasmid isolation techniques through the use of a spin miniprep kit. After plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to those of skill in the art.

[0228] The entry vector containing the sequence of SEQ ID NO: 82 was further recombined with the destination vector using LR Gateway recombinase. The destination vector used was pUCOE-DHFR-DEST, as described in Kaasboell et al., 2018, J. Biol. Chem, 293:46, pp. 17953-17970.

[0229] Competent E. coli (One Shot Top10) mutated to allow efficient propagation of plasmids TM After transfection of 1000 cells, QIAGEN TM The expression vector was isolated using standard plasmid isolation techniques using the Plasmid Plus Maxi Kit. The resulting expression vector was verified by standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to those skilled in the art. The resulting expression vector was then transformed into ExpiCHO TM "ExpiCHO" supplied by the manufacturer of Stable Production Medium (Gibco Cat. No. A3711001) TM The transfection was performed into ExpiCHO suspension culture-adapted CHO cells according to the "Generation and scale-up of stable cell lines using the product" protocol. Cells were maintained in vented Erlenmeyer flasks in a cell culture incubator maintained at 37°C with 8% CO2 on a shaker platform (as described in Kaasboell et al., supra). The transfected cells were cultured in ExpiCHO suspension culture. TM After overnight maintenance in expression medium, ExpiCHO cells were cultured in vitro supplemented with 0.1 μM methotrexate. TM The cells were then subcultured until viability approached 80%, at which point the medium was supplemented with 1 μM methotrexate. The cells were subcultured again until viability exceeded 95% and the doubling time decreased to less than 20 hours, at which point the cell pool was considered stably transfected. Once a stable cell pool was established, the cell culture volume was expanded and cultured for 1 *Stably transfected cells were allowed to be seeded for production at a density of 10^6 cells / mL. After 5 days, cells were pelleted by centrifugation at 4750xg, 4°C for 20 minutes, and the supernatant cell medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. Prior to chromatographic purification, 1M Na citrate pH 5.5 was added to a final concentration of 30 mM, and 2M L-arginine pH 4.0 was added to a final concentration of 100 mM.

[0230] A 5 mL HiTrap was prepared using the same protocol as described in Example 12, except that 100 mM L-arginine was added to buffers A1, A2, A3, and B1. TM MabSelect PrismA TM Capture step on column (GE Healthcare) followed immediately by 53mL HiPrep TM The protein was purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare). The purified protein preparation (not showing any signs of proteolytic processing) was then tested for its ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 10, the purified protein corresponding to SEQ ID NO: 80 produced from a stable transfection pool of CHO suspension cells surprisingly showed no signs of the ability to inhibit AKT phosphorylation (serine 473), and neither the intact dimeric Fc fusion protein preparation containing amino acids from CCN5 (SEQ ID NO: 41, Example 13, Figure 9) nor the intact dimeric Fc fusion protein preparation containing amino acids from CCN3 (SEQ ID NO: 80) was shown to be biologically active.

[0231] Example 15 An expression plasmid as described in Example 14 containing SEQ ID NO: 82, comprising amino acids 206 to 249 of CCN3 (SEQ ID NO: 44) in which amino acid 207 (isoleucine) is replaced by alanine, fused to the N-terminus of a peptide linker (SEQ ID NO: 22) and a chimeric Fc fragment of IgG subtype IgG2 / 4 (SEQ ID NO: 19), resulting in a protein sequence corresponding to SEQ ID NO: 80, to which is further added an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO: 32, and encoding a fusion protein corresponding to SEQ ID NO: 81, is transformed into an ExpiCHO plasmid as described in Example 9. TM Expression was achieved by transient transfection of cells. Six days after transfection, cells were pelleted as described in Example 14 and medium was replenished as described in Example 14. 5 mL HiTrap TM MabSelect PrismA TM Capture step on column (GE Healthcare) followed immediately by 53mL HiPrep TM The protein was purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare).

[0232] The purified protein preparations (partially proteolytically processed) were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 11, transiently transfected ExpiCHO TM Purified protein corresponding to SEQ ID NO:80 produced from cells exhibited a concentration-dependent ability to inhibit phosphorylation of AKT (serine 473), indicating that the expression system used to produce the fusion protein corresponding to SEQ ID NO:80, and consequently the degree of proteolytic processing observed, greatly influences the activity, or lack thereof, of the resulting protein preparation.

[0233] Example 16 A fusion protein comprising amino acids 206-249 (SEQ ID NO:44) of CCN3 with the amino acid at position 207 (isoleucine) substituted with alanine, fused to the N-terminus of a peptide linker (SEQ ID NO:21) and an Fc fragment (SEQ ID NO:55) containing monomer-inducing and half-life extending mutations, resulting in a protein sequence corresponding to SEQ ID NO:84, is added with an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO:32, resulting in a fusion protein corresponding to SEQ ID NO:85. The DNA sequence encoding the fusion protein of SEQ ID NO:85 was codon-optimized (according to the commercial supplier's algorithm) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID NO:86. Gateway attB sites were further added to both ends of the DNA sequence, resulting in a DNA sequence of SEQ ID NO:86. DNA sequences were synthesized and verified by commercial suppliers, then subcloned to generate plasmids as described in Example 9, and transformed into ExpiCHO vectors as described in Example 9. TM Expression was achieved by transient transfection of cells. Five days after transfection, cells were pelleted as described in Example 14, and medium was replenished as described in Example 14. 5 mL HiTrap TM MabSelect PrismA TM Capture step on column (GE Healthcare) followed immediately by 53mL HiPrep TM The protein was purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare).

[0234] Purified protein preparations exhibiting the expected monomeric form were subsequently tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 12, purified protein corresponding to SEQ ID NO: 84 exhibited a concentration-dependent ability to inhibit phosphorylation of AKT (serine-473), indicating that another monomeric fusion protein containing amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit phosphorylation of AKT (serine-473) in A549 human lung cancer cells.

[0235] Example 17 A fusion protein comprising amino acids 206-249 (SEQ ID NO:44) of CCN3 with the amino acid at position 207 (isoleucine) substituted with alanine, fused to the N-terminus of a peptide linker (SEQ ID NO:21) and an Fc fragment (SEQ ID NO:54) containing monomer-inducing and stability-inducing mutations, resulting in a protein sequence corresponding to SEQ ID NO:88, was added with an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO:32 to produce a fusion protein corresponding to SEQ ID NO:89. The DNA sequence encoding the fusion protein of SEQ ID NO:89 was codon-optimized (according to the algorithm of the commercial supplier) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced to the 3' end to produce a DNA sequence of SEQ ID NO:90. Gateway attB sites were further added to both ends of the DNA sequence to produce a DNA sequence of SEQ ID NO:91. The DNA sequence was synthesized and verified by the commercial supplier, and then subcloned to produce a plasmid as described in Example 9, and then transformed into an ExpiCHO vector as described in Example 9. TM Expression was achieved by transient transfection of cells. Six days after transfection, cells were pelleted as described in Example 14, and medium was replenished as described in Example 14. 5 mL HiTrap TM MabSelect PrismA TMCapture step on column (GE Healthcare) followed immediately by 53mL HiPrep TM The protein was purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare).

[0236] Purified protein preparations that predominantly exhibited the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 13, purified protein corresponding to SEQ ID NO: 88 exhibited a concentration-dependent ability to inhibit phosphorylation of AKT (serine-473), indicating that another monomeric fusion protein containing amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit phosphorylation of AKT (serine-473) in A549 human lung cancer cells.

[0237] Example 18 Peptide linker (SEQ ID NO: 93) and 6xHis tag, Halo tag and Sumo tag * A fusion protein containing amino acids 206 to 249 (SEQ ID NO: 44) of CCN3 with the amino acid at position 207 (isoleucine) replaced by alanine, fused to the N-terminus of a multifunctional tag containing elements (SEQ ID NO: 92), resulting in a protein sequence corresponding to SEQ ID NO: 94, was added with an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO: 32, resulting in a fusion protein corresponding to SEQ ID NO: 114. The DNA sequence encoding the fusion protein of SEQ ID NO: 114 was codon-optimized (according to the algorithm of the commercial supplier) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID NO: 95. Gateway attB sites were further added to both ends of the DNA sequence, resulting in a DNA sequence of SEQ ID NO: 96. The DNA sequence was synthesized and verified by the commercial supplier, and then subcloned to generate plasmids as described in Example 9, and ExpiCHO as described in Example 15.TM Expression was achieved by transient transfection of cells. Five days after transfection, cells were precipitated as described in Example 14. Prior to chromatographic purification, 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1 mM, 1M Na citrate pH 5.5 was added to a final concentration of 30 mM, 2M L-arginine pH 4.0 was added to a final concentration of 0.1 M, and imidazole was added to a final concentration of 5 mM.

[0238] 5mL HiTrap TM HisTrap TM Capture step on an Excel column (GE Healthcare) followed immediately by 53 mL HiPrep TM Proteins were purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare) and a FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell. TM The column was mounted on the first column switching valve. TM The column was loaded and equilibrated with A1 buffer consisting of 5 mM imidazole, 50 mM NaCl, 100 mM L-arginine, while the HiPrep TM The column was mounted on the second column switching valve and equilibrated with buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). TM With the second column switching valve containing the column set to bypass, 250 mL of harvested cell media containing the recombinant protein was pumped through the HisTrap column at a rate of 3.5 mL / min using the sample pump. TMAfter loading onto the column, it was washed with 5 column volumes of wash buffer A1, then 5 column volumes of wash buffer A3 (5 mM imidazole, 0.5 M NaCl, 100 mM L-arginine), then 2 column volumes of wash buffer A1. The HiPrep loaded onto the second column switching valve was then washed with elution buffer (250 mM imidazole, 50 mM NaCl, 100 mM L-arginine) before elution with elution buffer (250 mM imidazole, 50 mM NaCl, 100 mM L-arginine). TM The column was switched to enter the flow path. After elution with 10 mL of elution buffer, the HisTrap TM The column was switched out of the flow path and the purified protein was purified with Buffer A2 in HiPrep TM Protein elution was monitored by UV absorbance at 280 nm and collection was triggered when the absorbance exceeded 60 mAU.

[0239] Purified protein preparations exhibiting the expected monomeric form were subsequently tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 12, purified protein corresponding to SEQ ID NO: 94 exhibited a concentration-dependent ability to inhibit phosphorylation of AKT (serine-473), indicating that another monomeric fusion protein containing amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit phosphorylation of AKT (serine-473) in A549 human lung cancer cells.

[0240] Example 19 A fusion protein comprising amino acids 194 to 237 of CCN5 (SEQ ID NO:38) with the amino acid at position 195 (proline) substituted with alanine, fused to the N-terminus of a peptide linker (SEQ ID NO:21) and amino acids 25 to 609 of human serum albumin (SEQ ID NO:52), resulting in SEQ ID NO:97, was added with an N-terminal signal sequence for secretion resulting from human serum albumin of SEQ ID NO:32, resulting in a protein sequence corresponding to SEQ ID NO:98. The DNA sequence encoding the fusion protein of SEQ ID NO:98 was codon-optimized (according to the commercial supplier's algorithm) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end, resulting in the DNA sequence of SEQ ID NO:99. Gateway attB sites were further added to both ends of the DNA sequence, resulting in the DNA sequence of SEQ ID NO:100.

[0241] The DNA sequence was synthesized and verified by a commercial supplier and then subcloned to generate plasmids as described in Example 9, and DG44 CHO suspension cells engineered to express a constitutively active form of AKT were used to generate a stable pool of CHO suspension cells expressing the protein of SEQ ID NO:98 as described in Example 13. Once a stable cell pool was established, the cell culture volume was expanded to 1 * Stably transfected cells were allowed to be seeded for production at a density of 10^6 cells / mL. After 6 days, cells were pelleted by centrifugation at 4750xg for 20 minutes at 4°C, and the supernatant cell medium was harvested. Prior to chromatographic purification, 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1 mM, 0.5M EDTA was added to a final concentration of 2 mM, 1M Na citrate pH 5.5 was added to a final concentration of 30 mM, and 2M L-arginine pH 4.0 was added to a final concentration of 0.1M.

[0242] 3mL of CaptureSelect TMA 53 mL HiPrep immediately followed by a capture step on a Tricorn column (GE Healthcare) packed with human albumin affinity matrix (ThermoFisherScientific). TM Proteins were purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare) and a FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell. TM The column was mounted on the first column switching valve. TM The HiPrep column was loaded and equilibrated with A1 buffer consisting of 100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5. TM The column was mounted on the second column switching valve and equilibrated with Buffer A1 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). TM With the second column switching valve containing the column set to bypass, 500 mL of harvested cell media containing the recombinant protein was pumped through the CaptureSelect TM After loading onto the containing column, it was washed with 5 column volumes of wash buffer A1, then 5 column volumes of wash solution A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine, 0.25 M NaCl, pH 6.5), then 5 column volumes of wash buffer A1. The HiPrep loaded onto the second column switching valve was washed with elution buffer (30 mM citric acid, pH 3.5 + 0.5 M L-arginine) before elution with elution buffer (30 mM citric acid, pH 3.5 + 0.5 M L-arginine). TM The column was switched into the flow path. After elution with 10 mL of elution buffer, CaptureSelect TM The containing column was switched out of the flow path, and the purified protein was purified with Buffer A1 by HiPrep TM Protein elution was monitored by UV absorbance at 280 nm and collection was triggered when the absorbance exceeded 100 mAU.

[0243] Purified protein preparations exhibiting the expected monomeric form were subsequently tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 14, purified protein corresponding to SEQ ID NO: 97 exhibited a concentration-dependent ability to inhibit phosphorylation of AKT (serine-473), indicating that another monomeric fusion protein containing amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit phosphorylation of AKT (serine-473) in A549 human lung cancer cells.

[0244] Example 20 A fusion protein of human serum albumin (amino acids 25-606, SEQ ID NO:101) was fused at the C-terminus to a peptide linker (SEQ ID NO:22) linking amino acids 194-246 (SEQ ID NO:7) of human CCN5 in which the amino acid at position 195 (proline) was replaced by alanine, resulting in SEQ ID NO:103. To the fusion protein corresponding to SEQ ID NO:102, an N-terminal signal sequence for secretion originating from albumin of SEQ ID NO:32 was added to generate the fusion protein corresponding to SEQ ID NO:103. The DNA sequence encoding the fusion protein of SEQ ID NO:103 was codon-optimized (according to the algorithm of the commercial supplier) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end to generate the DNA sequence of SEQ ID NO:104. Gateway attB sites were further added to both ends of the DNA sequence to generate the DNA sequence of SEQ ID NO:105.

[0245] The DNA sequence was synthesized and verified by a commercial supplier and then subcloned to generate plasmids as described in Example 9, and DG44 CHO suspension cells engineered to express a constitutively active form of AKT were used to generate stable pools of CHO suspension cells expressing the protein of SEQ ID NO: 104 as described in Example 13. Once a stable cell pool was established, the cell culture volume was expanded to 1* Stably transfected cells were allowed to be seeded for production at a density of 10^6 cells / mL. After 6 days, cells were pelleted by centrifugation at 4750xg for 20 minutes at 4°C, and the supernatant cell medium was harvested. Prior to chromatographic purification, 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1 mM, 0.5M EDTA was added to a final concentration of 2 mM, 1M Na citrate pH 5.5 was added to a final concentration of 30 mM, and 2M L-arginine pH 4.0 was added to a final concentration of 0.1M.

[0246] A 3 mL CaptureSelect column was used as described in Example 19, except that the sample loading flow was 0.37 mL / min instead of 2.0 mL / min. TM A 53 mL HiPrep immediately followed by a capture step on a Tricorn column (GE Healthcare) packed with human albumin affinity matrix (ThermoFisherScientific). TM The protein was purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare).

[0247] Purified protein preparations showing the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 14, purified protein corresponding to SEQ ID NO: 102 showed a concentration-dependent ability to inhibit phosphorylation of AKT (serine-473), indicating that another monomeric fusion protein containing amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit phosphorylation of AKT (serine-473) in A549 human lung cancer cells.

[0248] Example 21 A fusion protein comprising amino acids 206-249 (SEQ ID NO:44) of CCN3, with the amino acid at position 207 (isoleucine) substituted with alanine, fused to the N-terminus of a peptide linker (SEQ ID NO:21) and amino acids 25-609 (SEQ ID NO:52) of human serum albumin, resulting in SEQ ID NO:106, was added with an N-terminal signal sequence for secretion resulting from albumin of SEQ ID NO:32, resulting in a fusion protein corresponding to SEQ ID NO:107. The DNA sequence encoding the fusion protein of SEQ ID NO:107 was codon-optimized (according to the algorithm of the commercial supplier) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end, resulting in the DNA sequence of SEQ ID NO:108. Gateway attB sites were further added to both ends of the DNA sequence, resulting in the DNA sequence of SEQ ID NO:109.

[0249] The DNA sequences were synthesized and verified by a commercial supplier and then subcloned to generate plasmids as described in Example 9, and transformed into ExpiCHO vectors as described in Example 9. TM Expression was achieved by transient transfection of cells. Six days after transfection, cells were pelleted as described in Example 14 and medium was replenished as described in Example 19. 10 mL of CaptureSelect TM A 53 mL HiPrep immediately followed by a capture step on a Tricorn column (GE Healthcare) packed with human albumin affinity matrix (ThermoFisherScientific). TM Proteins were purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare) and a FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell. TM The column was mounted on the first column switching valve. TMThe HiPrep column was loaded and equilibrated with A1 buffer consisting of 100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5. TM The column was loaded onto the second column switching valve and equilibrated with Buffer A1 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). TM With the second column switching valve containing the column set to bypass, 500 mL of harvested cell media containing the recombinant protein was pumped through the CaptureSelect TM After loading onto the containing column, it was washed with 3 column volumes of wash buffer A1, then 2 column volumes of wash buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine, 0.25 M NaCl, pH 6.5), then 3 column volumes of wash buffer A1. The HisPrep loaded onto the second column switching valve was washed with elution buffer (30 mM citric acid, pH 3.5 + 0.1 M L-arginine) before elution with elution buffer (30 mM citric acid, pH 3.5 + 0.1 M L-arginine). TM The column was set and entered into the flow path. After elution with 15 mL elution buffer, CaptureSelect TM The containing column was switched out of the flow path, and the purified protein was purified with Buffer A1 by HiPrep TM Protein elution was monitored by UV absorbance at 280 nm and collection was triggered when the absorbance exceeded 100 mAU.

[0250] Purified protein preparations containing the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 12, purified protein corresponding to SEQ ID NO: 106 showed a concentration-dependent ability to inhibit phosphorylation of AKT (serine-473), indicating that another monomeric fusion protein containing amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit phosphorylation of AKT (serine-473) in A549 human lung cancer cells.

[0251] Example 22 A fusion protein comprising amino acids 206-249 (SEQ ID NO:44) of CCN3, with the amino acid at position 207 (isoleucine) substituted with alanine, fused to the N-terminus of amino acids 25-609 (SEQ ID NO:52) of human serum albumin, resulting in SEQ ID NO:110, was added with an N-terminal signal sequence for secretion resulting from albumin, SEQ ID NO:32, resulting in a fusion protein corresponding to SEQ ID NO:111. The DNA sequence encoding the fusion protein, SEQ ID NO:111, was codon-optimized (according to the commercial supplier's algorithm) for protein expression in hamster cells, and a KOZAK sequence for translation was added to the 5' end and a stop codon was introduced at the 3' end, resulting in the DNA sequence, SEQ ID NO:112. Gateway attB sites were further added to both ends of the DNA sequence, resulting in the DNA sequence, SEQ ID NO:113.

[0252] DNA sequences were synthesized and verified by commercial suppliers, then subcloned to generate plasmids as described in Example 9, and transformed into ExpiCHO vectors as described in Example 9. TM Expression was achieved by transient transfection of cells. Six days after transfection, cells were pelleted as described in Example 14 and medium was replenished as described in Example 19. 10 mL of CaptureSelect TMA 53 mL HiPrep immediately followed by a capture step on a Tricorn column (GE Healthcare) packed with human albumin affinity matrix (ThermoFisherScientific). TM Proteins were purified by tandem chromatography consisting of desalting on a 26 / 10 desalting column (GE Healthcare) and a FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell. TM The column was mounted on the first column switching valve. TM The HiPrep column was loaded and equilibrated with A1 buffer consisting of 100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5. TM The column was mounted on the second column switching valve and equilibrated with Buffer A1 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). TM With the second column switching valve containing the column set to bypass, 300 mL of harvested cell media containing the recombinant protein was pumped through the CaptureSelect TM After loading onto the containing column, it was washed with 3 column volumes of wash buffer A1, then 2 column volumes of wash buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine, 0.25 M NaCl, pH 6.5), then 3 column volumes of wash buffer A1. The HiPrep loaded onto the second column switching valve was then washed with elution buffer (30 mM citric acid, pH 3.5 + 0.5 M L-arginine) before elution with elution buffer (30 mM citric acid, pH 3.5 + 0.5 M L-arginine). TM The column was set and entered into the flow path. After elution with 15 mL elution buffer, CaptureSelect TM The containing column was switched out of the flow path, and the purified protein was purified with Buffer A1 by HiPrep TM Protein elution was monitored by UV absorbance at 280 nm and collection was triggered when the absorbance exceeded 100 mAU.

[0253] Purified protein preparations containing the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Figure 15, purified protein corresponding to SEQ ID NO: 110 showed a concentration-dependent ability to inhibit phosphorylation of AKT (serine-473), indicating that another monomeric fusion protein containing amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit phosphorylation of AKT (serine-473) in A549 human lung cancer cells.

[0254] Example 23 The expression plasmid described in Example 21 containing SEQ ID NO: 108, which contains amino acids 206 to 249 (SEQ ID NO: 44) of CCN3, in which the amino acid at position 207 (isoleucine) is replaced by alanine, fused to the N-terminus of a peptide linker (SEQ ID NO: 21) and amino acids 25 to 609 (SEQ ID NO: 52) of human serum albumin, and further includes an N-terminal signal sequence for secretion originating from albumin of SEQ ID NO: 32, and which encodes a fusion protein corresponding to SEQ ID NO: 107, is used to perform stable transfection ExpiCHO as described in Example 14. TM A pool of cells was generated. The pool of stable transfected cells was expanded to produce a batch of conditioned medium containing the secreted protein corresponding to SEQ ID NO: 106. * Allowed seeding of stably transfected cells at a density of 10^6 cells / mL in a volume of 250mL. Cell culture was supplemented with 5% v / v 2XEfficientFeed TM C+(Gibco TM) was supplemented every other day starting 2 days after subculturing, and 2 days after subculturing, 3% glucose (10% w / v), and 6 days after subculturing, 5% glucose (10% w / v). After 9 days, cells were sedimented by centrifugation at 4750xg, 4°C for 20 min, and the supernatant cell medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. Prior to chromatographic purification, 1M Na citrate pH 5.5 was added to a final concentration of 30 mM, and 2M L-arginine pH 4.0 was added to a final concentration of 100 mM.

[0255] 10mL of CaptureSelect TM Proteins were purified by 2D chromatography consisting of a capture step on a Tricorn column (GE Healthcare) packed with human albumin affinity matrix (ThermoFisherScientific) followed immediately by size-exclusion chromatography on two consecutively coupled Superdex 200 Increase 10 / 300GL (GE Healthcare) columns. The FPLC chromatography system (BioRad NGC Discover) equipped with a 5 mm UV flow cell was used. TM The column was mounted on a 1000 sq. m. 10 Pro system, and the outlet valve was connected to a 5 mL sample loop. The CaptureSelect TM The column containing the recombinant protein was loaded onto the second column switching valve and equilibrated with buffer A1 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5), while the Superdex 200 Increase column was loaded onto the second column switching valve and equilibrated with buffer A1 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). With the second column switching valve containing the Superdex 200 Increase column set to bypass, 120 mL of harvested cell media containing the recombinant protein was pumped into the CaptureSelect TMThe harvested cell media containing the recombinant protein was loaded onto the CaptureSelect TM After loading onto the containing column, it was washed with 3 column volumes of Buffer A1, then 2 column volumes of Buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine, 0.25 M NaCl, pH 6.5), then 3 column volumes of Buffer A1. The CaptureSelect TM The containing column was eluted with 15 mL of Buffer B1 (30 mM citric acid, 0.5 M L-arginine, pH 3.5). TM After elution of the containing column, the CaptureSelect TM The containing column was switched out of the flow path and a second column switching valve was set to switch the Superdex 200 Increase containing column into the flow path. The CaptureSelect TM The eluate from the containing column was loaded onto a Superdex 200 Increase containing column in Buffer A1 at a rate of 0.5 mL / min. Protein elution was monitored by UV absorbance at 280 nm, and collection was triggered when the absorbance exceeded 200 mAU.

[0256] Purified protein preparations containing the expected monomeric form were subsequently tested for their ability to inhibit TGFβ-induced and active CCN2-induced activity in normal human lung fibroblasts (NHLFs) (Lonza Bioscience, Cat. No. CC-2512). NHLFs were subcultured in complete growth medium (Lonza Bioscience Bullet Kit (Cat. No. CC-3132) containing all additives (2% fetal bovine serum, insulin, hFGF-B, gentamicin / amphotericin B)) to maintain a density of up to 80% confluency according to the instructions of the commercial supplier (Lonza Bioscience). Active CCN2 is composed of domains 3-4 of CCN2 and was produced and purified as described in Kaasboell et al., 2018, supra.

[0257] To test the effect of the protein corresponding to SEQ ID NO: 106 on active CCN2 and TGFβ-induced cell migration of NHLFs (Transwell assay / modified Boyden chamber assay), cells were first detached with trypsin / EDTA, neutralized with trypsin neutralizing reagent (Lonza Bioscience, catalog number CC-5034), and resuspended in basal growth medium (fibroblast basal medium (Lonza Bioscience catalog number CC-3131, without any additives other than gensmycin (50 μg / mL))) before seeding 30,000 cells in a volume of 100 μL per well on the upper side of a transwell insert with a 5 μm pore size (24-well plates from SigmaAldrich (Merck KGaA), Corning® Transwell®, catalog number CLS3402-48EA). The lower chamber of the well contained the test substance or vehicle control dissolved in 500 μL of basal growth medium without any other additives other than gensmycin. After 20 hours of incubation, the inserts were removed from the wells and washed twice by immersion in phosphate-buffered saline (PBS, Lonza Bioscience, Cat. No. 17-512F) before being fixed in 4% formaldehyde (Solveco, Sweden, Cat. No. 621092) for 15 minutes at 37°C. The cells were permeabilized by treatment with 0.1% Triton X-100 in PBS for 10 minutes and then washed twice with PBS. The non-migrating cells on the upper side of the insert were scraped off with a cotton swap and the membrane was dried. The nuclei of the migrated cells on the upper side of the insert were stained with Hoechst 33342 20 mM (1:5000 dilution in PBS, ThermoFisherScientific, Cat. No. 62249) for 15 minutes in the dark and then washed twice by immersion in PBS. Cut the membrane from the transwell insert and mount it on a glass slide with the cells that have migrated towards the glass and add one drop of ProLong TMGold Antifade (ThermoFisherScientific, Cat. No. P36934), coverslips were mounted, and 5-10 images of each well were taken on a Zeiss Axio Observer Z.1 imaging system. Images were analyzed semi-automatically using ImageJ software v1.51k, Rasband, WS, Image J, National Institutes of Health, Bethesda, MD, USA, https: / / imagej.nih.gov / ij / , 1997-2018. As can be seen in Figure 16A, the protein corresponding to SEQ ID NO: 106 inhibits migration induced by both TGFβ and active CCN2.

[0258] To test the effect of the protein corresponding to SEQ ID NO: 106 on active CCN2 and TGFβ-induced scratch wound assays, NHLFs were detached with trypsin / EDTA and neutralized with trypsin neutralizing reagent (Lonza Bioscience, Cat. No. CC-5034) before seeding 100,000 cells in 1 mL volume in tissue culture treated 12-well plates (Corning Costar®, Cat. No. 3513). The day after seeding, cells were washed twice with 0.9% NaCl and the complete growth medium was replaced with basal growth medium. After incubation in basal growth medium for 16-20 hours, a scratch was made in the cell monolayer with a sterile 12.5 μL pipette tip (ThermoFisherScientific, Cat. No. 94420053) and cells were washed once with PBS before incubation in 1 mL of basal growth medium containing either the test substance or vehicle. Cells were further incubated for 24 hours, washed three times in PBS, and then fixed in 4% formaldehyde for 15 minutes at 37°C. After fixation, cells were washed again 3x3 minutes in PBS with gentle shaking and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes with gentle shaking. Cell nuclei were stained by treatment with Hoechst33342 20mM (1:5000 dilution in PBS, ThermoFisherScientific, Cat. No. 62249) for 15 minutes in the dark, followed by washing 3x5 minutes in PBS with gentle shaking. One drop of ProLong TM Gold Antifade (ThermoFisherScientific, Cat. No. P36934) was applied before mounting, and 5 images were captured from each well centered on the remaining gap on a Zeiss Axio Observer Z.1 imaging system. Images were analyzed by measuring the distance of the remaining gap after scratching at three fixed intervals along the length of the scratch wound. The average of all measurements from all images from each well was calculated and counted as one biological replicate. As can be seen from Figure 16B, the protein corresponding to SEQ ID NO: 106 inhibits the closure of scratch wounds induced by both TGFβ and active CCN2.

[0259] To test the effect of the protein corresponding to SEQ ID NO: 106 on TGFβ-induced gene regulation, NHLFs were detached with trypsin / EDTA and neutralized with trypsin neutralizing reagent (Lonza Bioscience, Cat. No. CC-5034) before seeding at 100,000 cells in a volume of 1 mL in tissue culture-treated 12-well plates (Corning Costar®, Cat. No. 3513). The day after seeding, cells were washed twice with 0.9% NaCl and the complete growth medium was replaced with 0.1% heat-inactivated fetal bovine serum (Gibco TM The wells were then replaced with basal growth medium supplemented with 0.1% fetal bovine serum (catalog no. 16000-044, heat inactivation performed as described in Example 2). After 6 hours of incubation in basal growth medium containing 0.1% fetal bovine serum, test substances or vehicle controls were added to the wells. After 96 hours, wells were washed twice with PBS and RNA was extracted using the Qiagen RNeasy RNA extraction kit (catalog no. 74106) according to the manufacturer's protocol. RNA concentrations were quantified with a NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, USA) and 200 ng RNA from each replicate was used to extract RNA using TaqMan PCR according to the manufacturer's protocol after dilution with nuclease-free water to a final RNA concentration of 50 ng / μL. TM cDNA was generated by utilizing the TaqMan reverse transcription kit (catalog number N8080234). TM Differential gene expression was analyzed from the resulting cDNA samples by TaqMan Fast Advanced Master Mix (ThermoFisherScientific, Cat. No. 4444557). Each sample was analyzed in technical triplicate using the Applied Biosystems StepOnePlus Real-Time PCR System according to the manufacturer's protocol. TMReal-time PCR reactions were performed. From the standard curves, the relative amounts of the different transcripts were calculated and then the technical triplicates were averaged to obtain a single value from each sample. All gene expression results were compared to GAPDH (ThermoFisherScientific, Cat. No. Hs02786624_gl) mRNA levels and normalized and expressed as fold of the mean of vehicle control stimulated wells. As can be seen in Figures 19C-F, the protein corresponding to SEQ ID NO:107 provides partial inhibition of TGFβ-induced genes that are generally considered to be pro-fibrotic genes: COL1A1 ("Collagen type 1 alpha-1", ThermoFisherScientific, Catalog No. Hs00164004_m1), FN1 ("Fibronectin 1", ThermoFisherScientific, Catalog No. Hs01549976_m1), ACTA2 ("Smooth muscle actin alpha-2", ThermoFisherScientific, Catalog No. Hs00426835_g1), and CCN2 (ThermoFisherScientific, Catalog No. Hs00170014_m1).

[0260] Overview of SEQ ID NOs referred to in the specification and sequence listing

[0261] [Table 2-1]

[0262] [Table 2-2]

[0263] [Table 2-3]

[0264] [Table 2-4]

[0265] [Table 2-5]

[0266] [Table 2-6]

[0267] [Table 2-7]

[0268] [Table 2-8]

[0269] [Table 2-9]

[0270] [Table 2-10]

[0271] [Table 2-11]

[0272] [Table 2-12]

[0273] Numbering of CCN proteins was according to the uniprot database as described in "Detailed Description of the Invention", supra. Numbering of Fc fragments was according to the Eu numbering system as described in "Detailed Description of the Invention", supra.

Claims

1. A monomeric fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110, and 111.

2. A DNA molecule encoding a monomeric fusion protein as defined in claim 1.

3. The DNA molecule of claim 2, further comprising a nucleotide sequence encoding a signal sequence.

4. A DNA molecule of claim 2 or 3, comprising a nucleotide sequence as set forth in SEQ ID NO: 86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113.

5. An expression vector comprising a DNA molecule as defined in any one of claims 2 to 4.

6. A host cell comprising a vector as defined in claim 5.

7. 13. A pharmaceutical composition comprising the fusion protein of claim 1 for use in therapy.

8. A pharmaceutical composition comprising the fusion protein of claim 1 for use in treating or preventing a disorder by inhibiting or counteracting cell signaling and cell physiological functions caused by a four-domain CCN family protein.

9. 9. A pharmaceutical composition according to claim 7 or 8 for use in the treatment or prevention of fibrosis, or a fibrotic condition or disease.

10. A pharmaceutical composition according to any one of claims 7 to 9 for use in the treatment of cancer.

Images